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Keywords:

  • FBI-1;
  • hepatocellular carcinoma;
  • prognosis;
  • chemotherapy;
  • 5-fluorouracil;
  • doxorubicin;
  • p53;
  • p21;
  • p27

Abstract

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. FUNDING SOURCES
  7. REFERENCES

BACKGROUND:

The so-called factor that binds to inducer of short transcripts-1 (FBI-1) purportedly plays an important role in tumorigenesis; however, its role in hepatocellular carcinoma (HCC) remains unknown. The objective of this study was to investigate the expression level, clinical relevance, and biologic function of FBI-1 in HCC.

METHODS:

Real-time quantitative reverse transcriptase-polymerase chain reaction analysis, Western blot analysis, and immunohistochemical staining were used to detect expression levels of FBI-1 and to analyze its relation to clinicopathologic parameters and to the prognosis of patients with HCC. In addition, the biologic functions of FBI-1 in regulating cell proliferation, migration, and reaction to chemotherapy were detected by using HepG2 cells and SMMC-7721 cells; subsequently, the molecular mechanism of FBI-1 also was investigated. Finally, a xenograft mouse model was used to validate the observations obtained from in vitro studies.

RESULTS:

Expression levels of FBI-1 messenger RNA and protein were elevated significantly in HCC tissues compared with adjacent nontumorous liver tissues (ANLTs). Increased FBI-1 expression was correlated with multiple tumor nodes, Edmondson-Steiner grade, and a poor prognosis in patients with HCC (P < .05). In vitro studies revealed that FBI-1 was capable of promoting cell proliferation (but not cell migration) by regulating the cell cycle regulation proteins p53, p21, and p27. In addition, FBI-1 could inhibit cell death induced by 5-fluorouracil or doxorubicin through suppressing the activation of p53. Consistent with the in vitro data, FBI-1 was capable of promoting cell proliferation and enhancing chemotherapy resistance of HCC in vivo.

CONCLUSIONS:

The current findings indicated that FBI-1 plays an important role in HCC carcinogenesis and chemotherapy tolerance, and FBI-1 may served as a novel prognostic marker and therapeutic target for HCC. Cancer 2012;. © 2011 American Cancer Society.

Hepatocellular carcinoma (HCC) is the sixth most common human malignancy worldwide, especially in China.1 With constantly increasing frequency, great numbers of new HCC diagnoses have been made every year (626,000).2 Because of its highly malignant potential, HCC ranks as the third leading cause of cancer death in the world, resulting in almost 600,000 deaths each year.2, 3 During the past decade, hepatic resection, which is the first treatment choice for HCC, has evolved into a safe procedure with an operative mortality rate <2% reported in some large clinical centers.4 However, with 5-year survival rates between 20% and 30% reported in the literature, the long-time survival of patients with HCC remains unsatisfactory because of the challenging problems in treating advanced and metastatic HCC.5, 6

Highlighting the mechanism that underlies the tumorigenesis of HCC is extremely important for its prevention and therapy. In recent years, investigators have achieved a better understanding of the molecular events leading to HCC oncogenesis. In parallel, multiple novel therapeutic agents are being assessed in HCC. There is accumulating evidence for the multistep nature of HCC.7 Epidemiological studies have revealed that many environmental risk factors, including chronic hepatitis B virus (HBV) and HCV infections, aflatoxin exposure, chronic alcohol abuse, cigarette smoking, and other factors are associated with the incidence of HCC.8, 9 In addition to environmental risk factors, genetic aberrations and molecular mechanisms involved in the development and progression of HCC have been uncovered in recent years, indicating their critical role in the initiation of hepatocarcinogenesis. For example, mutations in the p53, retinoblastoma (Rb), and/or insulin-like growth factor receptor 1 (IGFR1) genes have been reported in HCC,10 and the c-myc, locus at 8q24.12-13 frequently is up-regulated in HCC.11 Epidermal growth factor (EGF)-like domain 7 (Egfl7), which is overexpressed in HCC, may promote metastasis of HCC through regulating focal adhesion kinase (FAK) phosphorylation in an EGF receptor (EGFR)-dependent manner.12 Mutations of β-catenin commonly are observed in the early development of HCC, and disruption of this Wnt signaling protein affects the expression of its target genes, including c-myc, c-jun, cyclin D1, and fibronectin.13, 14 Work is ongoing to further identify novel, important HCC-associated proteins that may have profound effects as diagnostic or prognostic markers as well as molecular therapeutic targets to improve the outcome of patients with HCC.

FBI-1 (factor that binds to inducer of short transcripts-1) (also called leukemia/lymphoma-related factor [LRF]; osteoclast-derived zinc finger [OCZF]; Pokemon; and zinc finger and BR-C, ttk, and bab [BTB] domain-containing 7 [ZBTB7A]), which originally was purified as a transcription factor that binds to the bipartite inducer of the short transcripts element of human immunodeficiency virus, type 1, is a novel poxvirus and zinc finger (POZ)/BTB and Kruppel (POK) protein family member.15 POK proteins consist of an N-terminal POZ domain and a C-terminal Kruppel-type (C2H2) zinc finger domain. The C-terminal zinc fingers recognize and bind specific DNA sequences, and the POZ domain mediates homodimerization or heterodimerization and interacts with other proteins, such as corepressors, histone deacetylase, and other transcription factors, to regulate transcription.16-18 Previous studies have confirmed that FBI-1, as an important proto-oncogene, had multiple roles in carcinogenesis. Maeda et al reported that mouse embryo fibroblasts (MEFs) lacking FBI-1 were resistant to various oncogene-mediated transformation, and this resistance was restored when FBI-1 was coexpressed, suggesting a central role of FBI-1 in oncogenesis.19 The potential of FBI-1 as a genuine proto-oncogene also was examined in vivo in transgenic mice experiments in which mice with immature T-lymphoid and B-lymphoid cells that overexpressed FBI-1 developed thymic lymphomas and tumor infiltration of bone marrow.19 FBI-1 also enhances nuclear factor κB (NF-κB)-mediated transcription of E-selectin genes and overcomes gene repression by nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha (IκBα) or IκBβ through an interaction between the POZ domain of FBI-1 and the Rel homology domain (RHD) of NF-κB.20 In addition, FBI-1 can stimulate cell proliferation by repressing transcription of the tumor suppressors Rb, alternate reading frame (ARF), and p21.18, 21

FBI-1 is overexpressed in some human cancers, including lymphoma, nonsmall cell lung carcinoma, malignant gliomas, breast carcinoma, ect.19, 22-24 It is noteworthy that, despite the accumulation of evidence from in vitro and animal studies, to our knowledge, no analyses of any correlation between FBI-1 expression and clinicopathologic variables, patient prognosis, or the biologic function of FBI-1 in HCC have been reported to date. In the current study, we sought to determine expression levels of FBI-1 in HCC and their correlation with clinicopathologic characteristics and the prognosis of patients with HCC to explore its clinical value. Furthermore, the biologic function and molecular mechanism of FBI-1 in HCC carcinogenesis also were investigated both in vitro and in vivo.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. FUNDING SOURCES
  7. REFERENCES

Patients and Tissue Specimens

HCC tissue specimens were obtained from 129 patients with HCC who underwent hepatic resection at the Department of Surgery, Xiangya Hospital of Central South University between February 1998 and December 2005. These patients included 111 men and 18 women, and the median age was 45 years (range, 16-71 years). Specimens were paraffin embedded and stained with hematoxylin and eosin. Among the 129 HCC specimens, matched fresh specimens of HCC and adjacent nontumorous liver tissue (ANLT) specimens from 34 patients were collected and frozen in liquid nitrogen for detection with real-time quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) and Western blot analyses. Previous informed consent was obtained, and the study protocol was approved by the Ethics Committee of Xiangya Hospital of Central South University (Changsha City, People's Republic of China).

Real-Time qRT-PCR and RT-PCR

Real-time qRT-PCR and RT-PCR were performed as described previously.25 The following primers were used for real-time qRT-PCR: For FBI-1, the forward primer was 5′-TATGTCGCCAGATGCCAGGA-3′, and the reverse primer was 5′-GTCTCAGTGCAGCAGAGCGTCTA-3′; and, for glyceraldehyde 3-phosphate dehydrogenase (GAPDH), the forward primer was 5′-GCACCGTCAAGGCTGAGAAC-3′, and the reverse primer was 5′-TGGTGAAGACGCCAGTGGA-3′. The following primers were used for RT-PCR: For FBI-1, the forward primer was 5′-TGTGGGATAAACAGAGTCACGATC-3′, and the reverse primer was 5′-CAGAAGTGGATTCTACGTTTGTGGT-3′; and, for GAPDH, the forward primer was 5′-CTGCAGCATCTTCTCCTTCC-3′, and the reverse primer was 5′-CAAAGTTGTCATGGATGACC-3′. The results of real-time qRT-PCR were analyzed by using the 2− ΔΔCt method according to the formula ΔΔCt = (CtHCC-CtGAPDH) − (CtANLT − CtGAPDH).

Western Blot Analysis

Total protein was extracted and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then transferred onto polyvinylidene membranes (Millipore, Bedford, Mass). The blotted membranes were incubated with antihuman FBI-1 antibody (Santa Cruz Biotechnology, Santa Cruz, Calif) or with antihuman p27, p53, p21, and mouse double minute 2 (MDM2) antibodies (all from Santa Cruz Biotechnology) and then, in order, with secondary antibody (Santa Cruz Biotechnology). Beta-actin protein levels also were determined by using the specific antibody (Sigma, St. Louis, Mo) as a loading control.

Immunohistochemistry

Formalin-fixed paraffin sections (4 μm thick) were cut for immunohistochemical staining. After previous treatment as described elsewhere,12 the tissue sections were stained for FBI-1 (1:200 dilution; Santa Cruz Biotechnology) using the streptavidin-peroxidase system (Zhongshan Goldenbridge Biotechnology, Jiangsu, China). Negative control slides were probed with normal goat serum under the same experimental conditions. The immunohistochemical staining was scored by using the following 4-point scale according to the percentage of positive hepatocytes: 0 indicated <10% positive hepatocytes; 1+, 11% to 25% positive hepatocytes; 2+, 26% to 50% positive hepatocytes; and 3+, >51% positive hepatocytes.26 Thus, the levels of FBI-1 protein expression in HCC specimens were divided into a low expression group (with staining scores of 0 or 1+) and a high expression group (with staining scores of 2+ or 3+). Immunohistochemical analysis and scoring were performed by 2 independent investigators.

Follow-up and Prognostic Study

Follow-up data were obtained by direct communication with all 129 patients after they underwent hepatic resection. The follow-up period was calculated from the date of surgery to the date of either death or last follow-up. Recurrence and metastasis were diagnosed by clinical examination, serial α-fetoprotein (AFP) level measurement, and ultrasonography or computed tomography (CT) scans. Disease-free survival was defined as the duration of time that the patient survived after hepatectomy without signs of HCC. To determine the factors that influenced survival after surgery, the following 10 conventional variables, along with FBI-1 expression, were analyzed in all 129 patients: sex, age, preoperative AFP level, hepatitis history, cirrhosis, Child-Pugh score, tumor size, the number of tumor nodes, Edmondson-Steiner grade, and venous invasion.

Cell Lines and Cell Cultures

LO2, MHCC97-L, SMMC-7721, HCCLM3, MHCC97-H, Huh-7, and Bel-7404 cell lines were purchased from the Liver Cancer Institute of Fudan University (Shanghai, China). HepG2 and 293T cell lines were purchased from the American Type Culture Collection (ATCC) (Rockville, Md). These cell lines were cultured in high-glucose Dulbecco modified Eagle media (Corning Inc., Corning, NY), supplemented with 10% fetal bovine serum (GIBCO/Invitrogen, Carlsbad, Calif), and incubated in 5% CO2 at 37°C.

Plasmid Construction

FBI-1 whole-length combinational DNA (cDNA) was purchased from ATCC. The pBabe-flag-FBI-1 plasmid was prepared by cloning amplified cDNA fragments from FBI-1 whole-length cDNA and inserting it into the retrovirus plasmid pBabe (Addgene, Cambridge, Mass). The following oligonucleotide PCR primers were used: forward, 5′-CAGTGAATTCACCATGGACTACAAGGACGACGATGACAAGATGGCCGGCGGCGTGGACGGC-3′; reverse, 5′-CAGTGTCGACTTAGGCGAGTCCGGCTGTG AAGTTA-3′. In addition, the following small interfering RNAs (siRNAs) designed to knock down FBI-1 messenger RNA (mRNA) were purchased from Genechem (Shanghai, China): FBI-1, 5′-GCUGGACCUUGUAGAUCAAtt-3′ and 5′-UUGAUCUACAAGGUCCAGCtt-3′; negative control, 5′-CCUACGCCACCAAUUUCGUtt-3′ and 5′-ACGAAAUUGGUGGCGUAGGtt-3′.

Packaging Virus and Infection

The pBabe-flag-FBI-1 or pBabe control plasmid was transfected into 293T cells at 70% confluence together with packaging plasmid GAG-pol and pCMV-VSV-G by using Lipofectamine 2000 transfection reagent (Invitrogen). The retrovirus-containing supernatants were collected after 72 hours. For infection, the media containing retrovirus was added to the cells supplied with polybrene (8 μg/mL) for 6 hours and then replaced with fresh medium. Then, after 12 hours, the infection was repeated. Infected HepG2 cells were selected in the presence of puromycin (2 μg/mL) for 2 days, and puromycin-resistant cells were plated for further study. The stable cell lines were called HepG2+ FBI-1 cells and HepG2control cells. For FBI-1 knockdown, siRNA (500 pmol/35mm dish) was transfected into SMMC-7721 by using Lipofectamine 2000 reagent (Invitrogen). After 48 hours, the cells were harvested for further studies.

Growth Curves and Wound-Healing Assay

Methods for the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and wound-healing assays have been described previously.27, 28 These experiments were performed in triplicate.

Flow Cytometry

The cell cycle was analyzed by flow cytometry at different time points by using a propidium iodide (PI) cell cycle detection kit (Beyotime Institute of Biotechnology, Beijing, China). The cells were harvested, treated, and stained with PI according to the user's manual. The cell cycle was detected by using a flow cytometer (BD Company, Franklin Lakes, NJ).

Cell Viability Assay

Cell viability was detected with the AlamarBlue cell viability kit (Invitrogen, Carlsbad, Calif). First, cells were seeded in a 96-well cell culture plate (Corning Inc.) with 70% confluence, and the cells were cultured for 12 hours to allow them to adhere. Then, 5-fluorouracil (5-FU) or doxorubicin (Dox) was added to the medium with 1 μM final concentration separately. Twenty-four hours later, a 1:10 volume of AlamarBlue reagent was added directly to the cells in culture medium as described in the product manual. Then, the cells were incubated for 4 hours at 37°C in a cell culture incubator (protected from direct light). Cell viability was calculated by detecting the fluorescence intensity (FI) with a SpectraMax M5 plate reader (Molecular Devices, Sunnyvale, Calif). Cell viability as a percentage was expressed as follows: cell viability (%) = FItreatment/FIno treatment.

Dual-Luciferase Reporter Assay

Luciferase activity was assessed according to the Dual-Luciferase Reporter Assay protocol (Promega, Madison, Wis) using a Veritas 96-well Microplate Luminometer (Promega) with substrate dispenser (Promega). The p53 activity detection luciferase plasmid pGL3-p53 (firefly luciferase; Addgene) and control plasmid pRLTK (Renilla luciferase; Addgene) were cotransfected into HepG2+ FBI-1 and HepG2control cells. Twelve hours later, 5-FU or Dox was added to the medium to 1 μM final concentration, and the cells were incubated for another 24 hours. Then, the medium was removed, the cells were washed once with phosphate-buffered saline, and lysis buffer (50 μL) was added. A 10 μL sample was placed in each well of a 96-well plate; then, 50 μL Luciferase Assay Reagent II (a substrate for firefly luciferase) were added to each well by the luminometer, and the firefly activity was measured. Then, we added 50 μL Stop and Glow (a stop solution for firefly luciferase and a substrate for Renilla luciferase; the Stop and Glow reagent was included in the Dual-Luciferase Reporter Assay kit [Promega]), and Renilla luciferase activity was measured. The mean luciferase activity was measured and was used to calculate ratios between firefly and Renilla luciferase.

In Vivo Study

A xenografted mouse model of HCC was created by subcutaneous injection of 1 × 107 HepG2+ FBI-1 cells and HepG2control cells into the left upper flank regions of 5 experimental mice (male BALB/c mice, ages 3-4 weeks). For injection, the cells were resuspended in 0.2 mL Dulbecco modified Eagle media medium. The HepG2+ FBI-1 and HepG2control cell-xenografted mice were treated with 5-FU (30 mg/kg; 5 days of intraperitoneal injection and 2 days of withdrawal; 2 courses over 14 days) or Dox (1 mg/kg, orally administered once every 2 days for 18 days) starting on Day 20 after tumor implantation.29, 30 Then, the mice were killed; and the tumors were enucleated, fixed in 10% formalin, embedded in paraffin, and cut into 4 μm-thick slices for hematoxylin and eosin staining. The tumor size was calculated as follows: tumor volume (mm3) = (L × W2)/2, where L indicates the long axis, and W indicates the short axis. The relative tumor volume was calculated as follows: relative tumor volume (%) = (volume of tumor tissue treated with 5-FU or Dox)/(volume of tumor tissue treated without 5-FU or Dox). The Animal Use Committee of the Xiangya Hospital approved all protocols for treating animals.

Statistical Analysis

SPSS version 13.0 for Windows (SPSS Inc., Chicago, Ill) was used for statistical analyses. The Fisher exact test was used for statistical analysis of categorical data, and independent t tests were used to analyze continuous data. Survival curves were constructed using the Kaplan-Meier method and were evaluated using the log-rank test. In addition, a Cox proportional hazards regression model was established to identify factors that were associated independently with overall survival. All tests were 2-tailed, and P values <.05 were considered statistically significant.

RESULTS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. FUNDING SOURCES
  7. REFERENCES

FBI-1 mRNA and Protein Expression Levels Were Highly Elevated in HCC

Expression levels of FBI-1 mRNA were detected in 34 paired HCC tissues and ANLTs by real-time qRT-PCR analysis. HCC tissues expressed significantly higher FBI-1 mRNA levels than ANLTs, and the median elevated fold induction was 4.5-fold (range, 1.2-fold to 13.0-fold) (Fig. 1A). A classic semiquantitative RT-PCR analysis in parallel confirmed the real-time PCR results (HCC vs ANLT: 1.20 ± 0.28 vs 0.45 ± 0.12, respectively; P < .001) (Fig. 1B,C). To determine whether the difference in the mRNA levels was reflected at the protein level, we studies the same 34 paired specimens by using Western blot analysis. Consistent with the mRNA expression results, the FBI-1 protein expression levels also were significantly higher in HCC tissues than that in the corresponding ANLTs (0.95 ± 0.20 vs 0.31 ± 0.10; P < .001) (Fig. 1B,C).

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Figure 1. Messenger RNA (mRNA) and protein expression levels of factor that binds to inducer of short transcripts-1 (FBI-1) are were analyzed in samples of hepatocellular carcinoma (HCC) and adjacent nontumorous liver tissue (ANLT). (A) Real-time quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) results are shown from FBI-1 mRNA expression studies in 34 paired HCC tissues and ANLTs. Fold inductions were calculated using the formula 2− (ΔΔCT), and the median elevated fold induction was 4.5-fold (range, 1.2-fold to 13.0-fold). (B) FBI-1 mRNA and protein expression levels were detected by RT-PCR and Western blot analysis. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and β-actin were used separately as loading controls (bp indicates base pairs). (C) The abundance of FBI-1 mRNA and protein relative to the levels of loading controls was calculated. A single asterisk indicates a comparison of relative mRNA levels (1.20 ± 0.28 vs 0.45 ± 0.12; P < .001); double asterisk indicates a comparison of relative protein levels (0.95 ± 0.20 vs 0.31 ± 0.10; P < .001).

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Correlations Between FBI-1 Expression Levels and Clinicopathologic Parameters in HCC

The immunohistochemistry data from 129 HCC specimens were analyzed for the correlation of FBI-1 levels with clinicopathologic features. In 129 HCC samples, 49 samples (40%) scored 3+, 35 samples (27.1%) scored 2+, 28 samples (21.7%) scored 1+, and 17 samples (13.2%) scored 0. The representative positive immunostaining of HCC specimens is shown in Figure 2A-D. The correlations between FBI-1 protein staining intensity and clinicopathologic variables in patients with HCC were analyzed with the Mann-Whitney U test (see Table 1). The results indicated that the up-regulation of FBI-1 protein expression was correlated strongly with multiple tumor nodes (P = .035) and higher Edmondson-Steiner grade (P = .005). However, FBI-1 protein staining intensity was not related significantly to sex, age, preoperative AFP level, hepatitis history, Child-Pugh score, cirrhosis, tumor size, or venous invasion (P > .05) (Table 1).

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Figure 2. (A-D) Immunohistochemical staining and (E,F) survival analyses are shown for factor that binds to inducer of short transcripts-1 (FBI-1) expression in samples from 129 patients with hepatocellular carcinoma (HCC). In these representative images, nuclear expression is observed (A) in >51% of cancer cells (scored as 3+), (B) in 26% to 50% of cancer cells (scored as 2+), (C) in 11% to 25% of cancer cells (scored as 1+), and (D) in <10% of cancer cells (scored as 0+). On the basis of these immunohistochemical staining results, patients were classified according to the level of FBI-1 expression into a low expression group (scored as 0 or 1+; n = 45) and a high expression group (scored as 2+ or 3+; n = 84). (E,F) Log-rank tests indicated that patients with HCC in the high FBI-1 expression group had shorter overall survival and disease-free survival than patients in the low FBI-1 expression group.

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Table 1. Correlations Between FBI-1 Expression and Clinicopathologic Variables in 129 Patients With Hepatocellular Carcinoma
  FBI-1 Expression Level 
Clinicopathologic VariablesNo. of Patients0+1+2+3+Pa
  • Abbreviations: AFP, α-fetoprotein; FBI-1, factor that binds to inducer of short transcripts-1.

  • a

    Correlations between FBI-1 protein expression levels and clinicopathologic characteristics were evaluated using Spearman rank-correlation analysis.

Sex      
 Men11116223340 
 Women181629.048
Age, y      
 ≤6011011243243 
 >60196436.101
Preoperative AFP, ng/mL      
 ≤2054891720 
 >20759191829.092
Hepatitis history      
 Yes10914272840 
 No203179.339
Cirrhosis      
 Present8513202329 
 Absent44481220.140
Child-Pugh score      
 A10313232542 
 B2645107.819
Tumor size, cm      
 ≤5486131415 
 >58111152134.309
No. of tumor nodules      
 Multiple, ≥23317817 
 Solitary9616212732.035
Edmondson-Steiner grade      
 I-II7113182020 
 III-V584101529.005
Venous invasion      
 Present599131720 
 Absent708151829.391

Prognostic Implications of FBI-1 Expression

To examine the relation between FBI-1 expression levels and prognosis, 129 patients with HCC were divided according to immunohistochemical staining scores into a low FBI-1 expression group (score, 0 or 1+; n = 45) and a high FBI-1 expression group (score, 2+ or 3+; n = 84). The Kaplan-Meier method was used to analyze the correlation between FBI-1 expression levels and the prognosis of patients with HCC after hepatic resection. The results indicated that patients with high FBI-1 expression had shorter overall survival and disease-free survival than patients with low FBI-1 expression (median overall survival, 300 days vs 720 days; P = .008; median disease-free survival, 150 days vs 450 days; P = .002) (Fig. 2). To test whether the FBI-1 expression level was an independent prognostic factor for survival in patients with HCC, univariate and multivariate Cox regression analyses were used to identify factors that may predict survival after hepatic resection. The multivariate Cox regression analysis indicated that low FBI-1 expression (relative risk [RR], 0.42; P = .015), higher Edmondson-Steiner grade (RR, 0.40; P = .010), and the presence of venous invasion (RR, 3.33; P = .001) were independent prognostic factors for survival. The other clinicopathologic variables did not add any independent prognostic information (Table 2).

Table 2. Cox Regression Analyses of Overall Survival, Clinicopathologic Variables, and FBI-1 Expression
  Univariate AnalysisMultivariate Analysis
Clinicopathologic VariablesNo. of PatientsRR (95% CI)PaRR (95% CI)Pa
  • Abbreviations: CI, confidence interval; FBI-1, factor that binds to inducer of short transcripts-1; RR, relative risk.

  • a

    Statistically significant. Insignificant variables with P>.05 are not listed in the table, including sex, age, hepatitis history, serum α-fetoprotein level, cirrhosis, type of hepatectomy, Child-Pugh score, and margin status.

Tumor size, cm     
 ≤5481.00   
 >5812.70 (1.36-5.36).004
Edemondson-Steiner grade     
 I-II711.00 1.00 
 III-IV580.29 (0.16-0.54).0000.40 (0.20-0.80).010
Venous invasion     
 Absent701.00 1.00 
 Present595.07 (2.66-9.66).0003.33 (1.66-6.70).001
FBI-1 expression     
 High841.00 1.00 
 Low450.42 (0.21-0.82).0110.42 (0.21-0.84).015

FBI-1 Promotes Cell Proliferation but Not Migration of HCC In Vitro

To investigate the biologic function of FBI-1 in carcinogenesis and development of HCC, first, we detected FBI-1 expression levels in 7 HCC cell lines. The results indicated that, compared with the normal liver cell line LO2, HCC cells had higher FBI-1 expression levels, especially in MHCC97-L and SMMC-7721 cells (Fig. 3A). Then, we chose HepG2 cells and SMMC-7721 cells, which have different FBI-1 expression levels, for further studies.

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Figure 3. Factor that binds to inducer of short transcripts-1 (FBI-1) promotes cell proliferation but not cell migration in hepatocellular carcinoma (HCC) cells. (A) Endogenous expression levels of FBI-1 in 7 HCC cell lines were detected by Western blot analysis. The normal liver cell line LO2 was used as a loading control. (B) Growth curve data of HepG2+ FBI-1 cells and HepG2control cells indicated that HepG2+ FBI-1 cells grew much faster than HepG2control cells. An asterisk indicates P < .05; pound sign, P < .01. (C) Growth curve data from SMMC-7721control cells and SMMC-7721 short interfering RNA (siRNA) (SMMC-7721+ siRNA) cells indicate that SMMC-7721+ siRNA cells grew much slower than SMMC-7721control cells. An asterisk indicates P < .05; pound sign, P < .01. (D) A wound-healing assay indicates that there was no difference between HepG2+ FBI-1 cells, HepG2control cells, SMMC-7721control cells, and SMMC-7721+ siRNA cells after 24 hours. An ampersand indicates P > .05.

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We used the pBabe-flag-FBI-1 retrovirus and siRNA to up-regulate or knock down the expression of FBI-1 in HepG2 cells and SMMC-7721 cells separately. To characterize the biologic roles of FBI-1, growth curves were constructed and a wound-healing assay was performed as described above (see Materials and Methods). The results indicated that HepG2+ FBI-1 cells grew much faster than HepG2control cells (P < .05) (Fig. 3B), and FBI-1 knock down significantly inhibited SMMC-7721 cell proliferation (P < .05) (Fig. 3C). However, no significant difference in migration was observed between these cells (P > .05) (Fig. 3D).

FBI-1 Promotes Cell Proliferation by Down-Regulating p27, p53, and p21

To highlight the potential mechanism by which FBI-1 regulates HCC cell proliferation, the cell cycle distribution of these cells was detected by flow cytometry. The results indicated that FBI-1 overexpression could dramatically increase G2/M phase cell numbers (14.5% vs 1.3%; P < .05) (Fig. 4A), and FBI-1 knock down consistently decreased G2/M phase cell numbers (12% vs 22%; P < .05) (Fig. 4B). On the basis of the finding that FBI-1 could regulate cell cycle progression, the expression levels of some cell cycle-related proteins were detected by Western blot analysis. We observed that HepG2+ FBI-1 cells and SMMC-7721control cells had significantly lower expression levels of p27, p53, and p21 than the corresponding HepG2+ FBI-1 cells and SMMC-7721+ siRNA cells, suggesting an inverse correlation between FBI-1 and p27, p53, and p21 (Fig. 4C). These data indicated that FBI-1-regulated cell proliferation may be controlled through p27, p53 and p21.

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Figure 4. Factor that binds to inducer of short transcripts-1 (FBI-1) promotes cell proliferation by repressing cell cycle-related protein p27, p53, and p21 expression. (A) Cell cycle distribution of HepG2+ FBI-1 cells and HepG2control cells was detected by flow cytometry. (B) Cell cycle distributions of HepG2+ FBI-1 cells and HepG2control cells are illustrated. (C) Expression levels of p27, p53, and p21 were detected by Western blot analysis.

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FBI-1 Inhibits 5-FU-Induced or Dox-Induced Cell Death by Suppressing the Activation of p53

Because FBI-1 was able to regulate the expression of p53, which plays a critical role in cell death, we hypothesized that FBI-1 might play an important role in chemotherapy. To verify this possibility, cells were treated with the most commonly used chemotherapy reagents—5-FU (1 μM) or Dox (1 μM)—and cell viability was detected by using the AlamarBlue Cell Viability Kit (Invitrogen). The results indicated that HepG2+ FBI-1 cells were much more resistant to 5-FU-induced or Dox-induced cell death (5-FU treatment group: 0.75 ± 0.13 vs 0.53 ± 0.10; P < .05; Dox treatment group: 0.57 ± 0.11 vs 0.25 ± 0.05; P < .05) (Fig. 5A), and SMMC-7721+ siRNA cells were much more sensitive to 5-FU-induced or Dox-induced cell death (5-FU treatment group: 035 ± 0.08 vs 0.60 ± 0.12; P < .05; Dox treatment group: 0.20 ± 0.04 vs 0.45 ± 0.07; P < .05) (Fig. 5B).

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Figure 5. Factor that binds to inducer of short transcripts-1 (FBI-1) inhibited 5-fluorouracil (5-FU)-induced and doxorubicin (Dox)-induced cell death in hepatocellular carcinoma cells by suppressing the activation of p53. The cells were treated with 5-FU (1 μM) or Dox (1 μM) for 24 hours. Then, cell viability was detected by using the AlamarBlue assay (Invitrogen, Carlsbad, Calif). (A) This chart illustrates the viability of HepG2+ FBI-1 cells and HepG2control cells. (B) This chart illustrates the viability of SMMC-7721control cells and SMMC-7721+ siRNA cells. (C) The activity of p53 and its changes after 5-FU (1 μM) or Dox (1 μM) treatment in HepG2+ FBI-1 and HepG2control cells were detected by using a dual-luciferase reporter assay. A single asterisk indicates P < .01; double asterisks, P < .05; triple asterisks, P < .01; a single pound sign, P > .05; double pound signs, P > .05. (D) Expression levels of p53 in HepG2+ FBI-1 and HepG2control cells before and after 5-FU or Dox treatment were detected by Western blot analysis. MDM2 indicates mouse double minute 2.

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Furthermore, dual-luciferase reporter assay results indicated that the activation level of p53 in HepG2+ FBI-1 cells was significantly lower than that in HepG2control cells (0.38 ± 0.10 vs 0.99 ± 0.15; P < .01); and, after 5-FU or Dox treatment, p53 was activated significantly in HepG2control cells (5-FU treatment: 1.95 ± 0.35 vs 0.99 ± 0.15; P < .05; Dox treatment: 2.1 ± 0.37 vs 0.99 ± 0.15; P < .01). However, the activation of p53 was repressed significantly in HepG2+ FBI-1 cells (5-FU treatment: 0.48 ± 0.12 vs 0.38 ± 0.10; P > .05; Dox treatment: 0.73 ± 0.18 vs 0.38 ± 0.10; P > .05) (Fig. 5C). Consistent with the luciferase data, Western blot results also confirmed that FBI-1 could decreased the expression of p53 and inhibited its activation after 5-FU or Dox treatment (Fig. 5D).

FBI-1 Promotes Cell Proliferation and Enhances Chemotherapy Resistance of HCC In Vivo

To validate the observations obtained from in vitro studies, we examined the in vivo relevance of the potential role for FBI-1 in HCC tumorigenesis and chemotherapy in a xenografted mouse model. We observed that the average size of tumors in HepG2+ FBI-1 mice was dramatically larger than those in HepG2control mice (1485 ± 350 mm3 vs 648 ± 130 mm3; P < .05), and tumors in the HepG2+ FBI-1 mice were much more resistant to 5-FU or Dox treatment than those in the HepG2control mice (relative tumor volume: 5-FU treatment, 40% vs 24%; P < .05; Dox treatment, 27% vs 10%; P < .05) (Fig. 6A). Hematoxylin and eosin staining also revealed that tumors in HepG2control mice had more cell death than those in HepG2+ FBI-1 mice (Fig. 6B).

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Figure 6. Factor that binds to inducer of short transcripts-1 (FBI-1) promotes cell proliferation and enhances chemotherapy resistance of hepatocellular carcinoma in vivo. (A) (Top) Tumors from the HepG2+ FBI-1 and HepG2control groups treated with or without 5-fluorouracil (5-FU) or doxorubicin (Dox) are shown. (Bottom Left) Tumor size was dramatically larger in the HepG2+ FBI-1 group than in the HepG2control group (single asterisk; P < .05). (Bottom Right) Tumors in the HepG2+ FBI-1 group were much more resistant to 5-FU or Dox treatment than tumors in the HepG2control group (single asterisk indicates the 5-FU treatment group, P < .05; double asterisks, the Dox treatment group; P < .05. The volume of tumors was calculated as follows: tumor volume (mm3) = (length × width2)/2, relative tumor volume = (volume of tumor tissue treated with 5-FU or Dox)/(volume of tumor tissue treated without 5-FU or Dox). (B) Hematoxylin and eosin staining was used in the HepG2+ FBI-1 and HepG2control groups that did and did not receive treatment with 5-FU or Dox (original magnification, ×400).

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DISCUSSION

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. FUNDING SOURCES
  7. REFERENCES

Recently, many studies have confirmed that FBI-1 is an important proto-oncogene and is deregulated in many cancers. Apostolopoulou et al observed prominent expression of FBI-1 in nonsmall cell lung cancerous areas compared with adjacent normal tissue elements and noted that this was caused by gene amplification (2-fold to 5-fold).22 Another study also indicated that, by comparing the expression levels of FBI-1 in 20 human benign and malignant breast biopsy tissues, the overall expression of FBI-1 mRNA transcripts and total protein was significantly more in malignant breast tissues than in benign breast tissues.24 However, the correlation between its expression and clinicopathologic parameters or the prognosis of patients has not been documented, and the expression profile and function of FBI-1 in HCC currently remain unknown. Given the accumulating evidence and information, we hypothesized that FBI-1 also plays an important role in HCC carcinogenesis. To clarify this hypothesis, the current study was performed to investigate the expression, clinical relevance, and biologic role of FBI-1 in HCC.

In this study, first, we detected FBI-1 expression profiles in HCC; then, we analyzed the correlations of its expression with clinicopathologic parameters and prognosis in patients with HCC. Our data revealed that both mRNA and protein levels of FBI-1 were highly elevated in HCC tissues compared with levels in the corresponding ANLTs. In addition, the up-regulation of FBI-1 protein expression was strongly correlated with multiple tumor nodes and higher Edmondson-Steiner grade, but not with other clinicopathologic variables. On the basis of the well known common sense that multiple tumor nodes and poor cell differentiation are highly correlated with a poor prognosis in patients with HCC,31, 32 the value of FBI-1 in predicting the outcome of patients with HCC after hepatic resection also was evaluated. We observed that up-regulated expression of FBI-1 was correlated with shorter overall and tumor-free survival in patients with HCC. It is noteworthy that the multivariate Cox regression analysis indicated that FBI-1 expression, together with Edmondson-Steiner grade and venous invasion, was an independent prognostic factor for survival. These data suggest that FBI-1 expression may serve as a novel prognostic marker in patients with HCC.

Because of its valuable clinical relevance, we believed that FBI-1 plays an important role in the development and progression of HCC, which subsequently was verified in our in vitro and in vivo studies. Our data indicated that FBI-1 can regulate the proliferation, but not the migration, of HCC cells through p27, p53, and p21, which are involved in the initiation and development of HCC.33, 34 p27Kip1 is a member of the CIP/KIP family that can cause G1 cell cycle arrest by inhibiting the activities of G1 cyclin–cyclin-dependent kinases. Thus, as a negative regulator of the cell cycle, p27Kip1 is overexpressed in the early stages of hepatocarcinogenesis, indicating that this parameter may be a useful diagnostic marker for precancerous lesions and early HCC.33 In the current study, we confirmed that FBI-1 is capable of decreasing p27 expression, suggesting that FBI-1 up-regulation may be an early event in HCC carcinogenesis. The tumor suppressor p53, which is a key regulator of cell proliferation and apoptosis, is always inactivated or mutated in human cancers.35, 36 However, Teramoto et al observed that p53 mutation is a late event and occurs in only 30% of HCC grade III tissues.37 So, we image that FBI-1, together with other p53 regulation networks, can lead to the inactivation of p53 in early stage HCC. The cyclin-dependent kinase inhibitor p21 is regulated by p53-dependent and p53-independent pathways. Choi et al observed that FBI-1 acts as a transcriptional repressor of p21 by binding to the distal p53-responsive elements of the p21 promoter,18 and we believe that further investigations are needed to determine whether the down-regulation of p21 by FBI-1 is p53-dependent or p53-independent. FBI-1 also can repress the expression of ARF and Rb by directly binding to their promoter; however, no distinct change was observed in our study. This may be because of differences in the caner type.

In the current study, we also investigated the role of FBI-1 in chemotherapy. The results indicated that FBI-1 can inhibit cell death induced by 5-FU or Dox by suppressing the activation of p53. Finally, our in vivo studies of a mouse xenograft model validated the observations obtained from our in vitro studies. Consistent with the in vitro results, FBI-1 was able to promote cell proliferation and enhance chemotherapy resistance to 5-FU or Dox in vivo. On the basis of its biologic function, these results indicated that suppressing the expression of FBI-1 can inhibit cell proliferation and increase the sensitivity of HCC to chemotherapy, which can serve as a new therapeutic strategy to treat HCC. Indeed, a recent study indicated that curcumin may inhibit cell proliferation in human lung cancer cells by decreasing the expression of FBI-1.38

In summary, the current study demonstrated that proto-oncogene FBI-1 levels were highly elevated in HCC, and the overexpression of FBI-1 was correlated with a poor prognosis in patients with HCC, promoted cell proliferation, and enhanced chemotherapy resistance of HCC in vitro and in vivo. These results indicate that FBI-1 may serve as a novel prognostic marker and a therapeutic target for HCC.

FUNDING SOURCES

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. FUNDING SOURCES
  7. REFERENCES

This work was supported by grants from the National Key Technologies R&D Program of China (grants 2001BA703B04 and 2004BA703B02), the National Keystone Basic Research Program of China (grant 2004CB720303), the National High Technology Research and Development Program of China (grant 2006AA02Z4B2), the National Science Fund for Distinguished Young Scholars of China (grant 30328028), the National Natural Science Foundation of China (grant 30571826), the Clinical Subjects' Key Project of Ministry of Health (2007-2009), and the National Science and Technology Major Projects (grant 2009ZX09103-681).

CONFLICT OF INTEREST DISCLOSURES

The authors made no disclosures.

REFERENCES

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. FUNDING SOURCES
  7. REFERENCES