Far upstream element-binding protein 1 is a prognostic biomarker and promotes nasopharyngeal carcinoma progression

Abstract

Nasopharyngeal carcinoma (NPC) is a malignant epithelial tumor with tremendous invasion and metastasis capacities, and it has a high incidence in southeast Asia and southern China. Previous studies identified that far upstream element-binding protein 1 (FBP1), a transcriptional regulator of c-Myc that is one of the most frequently aberrantly expressed oncogenes in various human cancers, including NPC, is an important biomarker for many cancers. Our study aimed to investigate the expression and function of FBP1 in human NPC. Quantitative real-time RT-PCR (qRT-PCR), western blot and immunohistochemical staining (IHC) were performed in NPC cells and biopsies. Furthermore, the effect of FBP1 knockdown on cell proliferation, colony formation, side population tests and tumorigenesis in nude mice were measured by MTT, clonogenicity analysis, flow cytometry and a xenograft model, respectively. The results showed that the mRNA and protein levels of FBP1, which are positively correlated with c-Myc expression, were substantially higher in NPC than that in nasopharyngeal epithelial cells. IHC revealed that the patients with high FBP1 expression had a significantly poorer prognosis compared with the patients with low expression (P=0.020). In univariate analysis, high FBP1 and c-Myc expression predicted poorer overall survival (OS) and poorer progression-free survival. Multivariate analysis indicated that high FBP1 and c-Myc expression were independent prognostic markers. Knockdown of FBP1 reduced cell proliferation, clonogenicity and the ratio of side populations, as well as tumorigenesis in nude mice. These data indicate that FBP1 expression, which is closely correlated with c-Myc expression, is an independent prognostic factor and promotes NPC progression. Our results suggest that FBP1 can not only serve as a useful prognostic biomarker for NPC but also as a potential therapeutic target for NPC patients.

Figure 1

The expression of FBP1 directly correlates with c-Myc in nasopharyngeal carcinoma. (a) Western blots of normal human nasopharyngeal epithelial cell samples (N03 and N06) and samples of nasopharyngeal carcinoma cell lines using antibodies against FBP1 and c-Myc (left panel). The relationship between FBP1 and c-Myc is shown in the right panel. β-Actin was used as a control for protein loading and integrity. (b) FBP1 and c-Myc mRNA levels in a series of NPC cell lines compared with the normal human nasopharyngeal epithelial cells. mRNA levels are presented as the means±S.D. and are normalized to the housekeeping gene β-actin using qRT-PCR. (c) The relative expression of FBP1 and c-Myc in 29 pairs of matched NPC and non-tumor tissues at the transcript level (left and middle panels). The right panel shows the association between FBP1 and c-Myc

Figure 2

FBP1 silencing suppressed the proliferation and clone formation of NPC cells. (a, b) Western blot samples from CNE2 (a) and 5-8F (b) cells were transfected with NC- or FBP-targeting (KD) siRNAs. β-Actin was used as an internal control (upper panel). The MTT assay measured the viability of CNE2 and 5-8F cells with NC- or FBP1-targeting (KD) siRNAs. The data represent the means±S.E.M. (lower panel; **P<0.01, ***P<0.001 by the paired t-test). (c, d) The representative pictures (c) and the quantitative analysis (d) of colony-formation assay of CNE2 and 5-8F cells transfected with NC- or FBP1-targeting (KD) siRNAs. The assay was performed in triplicate. The data represent the means± S.E.M. (**P<0.01, ***P<0.001 by the paired t-test)

Figure 3

Knockdown of FBP1 decreased the side population in NPC cells. (a) Flow cytometry analysis of Hoechst 33342 staining was performed in CNE2 cells transfected with NC- or FBP1-targeting (KD) siRNAs (upper panel) and treated with ko143 (final concentration: 5 μm) before Hoechst 33342 staining (lower panel). The percentage of SP cells is indicated. (b) Flow cytometry analysis of Hoechst 33342 staining was performed and 5-8F cells transfected with NC- or FBP1-targeting (KD) siRNAs (upper panel) and treated with ko143 (final concentration: 5 μm) before Hoechst 33342 staining (lower panel). The percentage of SP cells is indicated. (c) Representative western blots of FBP1, c-Myc, ABCG2 and β-actin in CNE2 and 5-8F cells transfected with NC- or FBP1-targeting (KD) siRNAs. β-Actin served as a loading control

Figure 4

Results of radiation and chemotherapy sensitivity. (a) The growth curves of CNE2 cells transfected with NC- or FBP1-targeting (KD) siRNAs at 48 h post treatment with the indicated doses of DDP (left panel) and 5-FU (right panel). (CNE2-NC versus CNE2-siFBP1-1#1 and siFBP1-2#, P<0.001 by the paired t-test). (b) The growth curves of 5-8F cells transfected with NC- or FBP1-targeting (KD) siRNAs at 48 h post treatment with the indicated doses of DDP (left panel) and 5-FU (right panel). (5-8F-NC versus 5-8F-siFBP1-1# and 5-8F-siFBP1-2#, P<0.05, by the paired t-test). (c) The survival fraction (SF) curves (upper panel) and the representative pictures of CNE2 and 5-8F cells transfected with NC- or FBP1-targeting (KD) siRNAs at 10 days after X-ray irradiation with the indicated Gy doses (lower panel). (NC versus siFBP1-1#1 in CNE2 and 5-8F, P<0.05; NC versus siFBP1-2# in CNE2 and 5-8F, P<0.01 by the paired t-test)

Figure 5

The expression of FBP1 in nasopharyngeal carcinoma (NPC) by immunohistochemical (IHC) staining Positive expression of FBP1 was primarily detected in the nucleus of the NPC cells. (a) Negative expression of FBP1 in normal nasopharyngeal tissues. (SP x400, left upper panel). (b) Moderate expression of FBP1 in NPC. (SP x400, right upper panel). (c) High expression of FBP1 in NPC. (SP x400, right lower panel). (d) Negative expression of c-Myc in normal nasopharyngeal tissues. (SP x400, left upper panel). (e) Moderate expression of c-Myc in NPC. (SP x400, right upper panel). (f) High expression of c-Myc in NPC. (SP x400, right lower panel). (g) The correlation of FBP1 and c-Myc expression by IHC

Figure 6

Relationship between FBP1 and patient survival. (a) Overall survival curves and progression-free survival curves of patients with low and high FBP1 expression. (b) Overall survival curves and progression-free survival curves of patients with high expression of both FBP1/c-Myc, low expression of both FBP1/c-Myc and high expression of either FBP1 or c-Myc

Figure 7

FBP1 silencing reduced the tumorigenicity of NPC cells. (a) Images of the tumors formed by CNE2 cells transfected with NC or siFBP1. (b) The growth curve of tumors formed by CNE2 cells transfected with NC or siFBP1. The data are presented as the means±S.D. (n=6 mice in each group). (c–d) HE staining pictures of mice treated with NC (c) or with siFBP1 (d) (SP x400). (e–f) IHC staining pictures of FBP1 expression in the primary xenografts from cells treated with NC (e) or with siFBP1 (f) (SP x400). (g–h) IHC staining pictures of c-Myc expression in the primary xenografts from cells treated with NC (g) or with siFBP1 (h) (SP x400)