Hepatocellular carcinoma (HCC) is a highly lethal cancer whose prognosis is typically poor. It ranks the third cause for cancer deaths in East Asia and sub-Saharan Africa,1, 2 and the second for male cancer deaths in China.3 Now, its incidence is also increasing in the United States and Europe.4, 5 HCC generally carries a poor prognosis, with its 5-year survival rate as low as 25–39% after surgery.6 The majority of HCC patients (>80%) present with an advanced stage for which chemotherapy and radiotherapy have limited efficacy.7 Even for those with their tumor resected, the recurrence rate can be as high as 50% at 2 years.8, 9 Several clinicopathological parameters, including poorly differentiated phenotype, portal venous invasion and intrahepatic metastasis, have been found to contribute to poor prognosis of HCC. However, the mechanisms underlying the development of HCC remain unclear.
Gene amplification and consequent overexpression of many oncogenes, such as c-myc, cyclin D1, CREB3L4 (cAMP responsive element binding protein 3-like 4), INTS3 (integrator complex subunit 3), SNAPAP (SNAP-associated protein), ALC1 (Amplified in Liver Cancer 1),10–12 have been detected frequently in various types of solid tumors, including HCC. They may play an important role in tumor pathogenesis, probably because the overexpression of these oncogenes confers a growth advantage.13–16 Small guanosine triphosphate (GTP)-binding proteins of the Ras superfamily act as molecular switches in many biological processes.17, 18 Mutations or deregulation of these proteins can contribute to malignant phenotypes of human tumors. Guanine nucleotide exchange factors (GEFs), which catalyze the dissociation of guanosine diphosphate from inactive GTP-binding proteins, positively regulate these GTP-binding proteins in response to a variety of signals. Currently, many GEFs, including Vav1, LARG, Bcr and T-lymphoma invasion and metastasis 1 (Tiam1), have been identified as oncogenes.19–24 Tiam1 is a metastasis-related gene of T lymphoma and also involved in the metastasis of a variety of cancers.25 It was first identified as a gene which might induce invasion and metastasis by proviral tagging, in combination with in vitro selection for invasiveness in T lymphoma cells.26 The role of Tiam1 in cellular migration, invasion and metastasis may not be limited to T lymphoma. It has been reported to be significant in promoting tumor progression in a variety of cancers, such as breast cancer, colorectal cancer (CRC), lung cancer and Ras-induced skin tumors.27–30 Tiam1 displays GEF activity toward 3 Rho-like GTPases, Rac1, Cdc42 and RhoA in vitro, but it specifically activates Rac in vivo.31 Increasing evidences suggest that Tiam1 may not only promote the activation of Rac GTPases but also influence their signaling specificity.32, 33 It directly binds to many different cytoplasmic and membrane-associated proteins, coupling Tiam1-Rac activity to specific signaling pathways.34
Recently, Engers et al. has suggested that Tiam1 overexpression in prostate cancer might be a new and independent predictor of poor prognosis for patients with prostate cancer.35 However, the potential prognostic relevance of Tiam1 expression in HCC has not yet been investigated.
In this study, we attempted to investigate the expression of Tiam1 in primary HCC using immunohistochemical analysis, identify its relationship with clinicopathological features and evaluate its prognostic value in respect of survival in HCC patients.
Material and methods
Patients and tissue samples
A total 152 patients with primary HCC, who had undergone routine surgery at Nangfang Hospital and Zhujiang Hospital, Guangzhou City, Guangdong Province, China between 1999 and 2002, were enrolled in this study. They were not pretreated with radiotherapy or chemotherapy prior to surgery. They were followed up for 5 years and their complete clinical data were collected. All primary HCC samples taken from the 152 patients were fixed in 10% formalin, embedded in paraffin, sectioned consecutively at 4 μm and stained by hematoxylin and eosin. The histological types were assigned according to the criteria of the WHO classification system by 3 pathologists independently in a double-blinded manner.
Cell lines preparation
A normal hepatocyte line (HL-7702) was purchased from the cell bank of Chinese Academy of Sciences and maintained in RPMI 1640 (Hyclone) supplemented with 20% fetal bovine serum (FBS). The HCC cell lines, Hep3B, HepG2, BEL-7402, BEL-7404, BEL-7405, SMMC-7721, QGy-7701, QGy-7703, were purchased also from the cell bank of Chinese Academy of Sciences and maintained in RPMI 1640 (Hyclone) supplemented with 10% FBS. All the cell lines were in a 5% CO2 humidified atmosphere at 37°C.
The sections were deparaffinized and rehydrated, and endogenous peroxidase was inhibited with 0.3% H2O2 methanol. For antigen retrieval, slides were boiled in 0.01 M, pH 6.0 sodium citrate buffer for 15 min in a microwave oven. After blocked with the 5% normal goat serum, primary antibody (rabbit anti-Tiam1, Santa Cruz Biotechnology, Santa Cruz, CA) in blocking buffer (1:100) was applied and the slides were incubated at 4°C overnight. Following incubation with biotinylated secondary antisera, the streptavidin–biotin complex/horseradish peroxidase was applied. Finally, the visualization signal was developed with 3,3′-diaminobenzidine tetrahydrochloride, and the slides were counterstained in hematoxylin.
The total Tiam1 immunostaining score was calculated as the sum of the percent positivity of stained tumor cells and the staining intensity. The percent positivity was scored as “0,” 0%; “1,” 1–10%; “2,” 11–50%; “3,” 51–80% and “4,” >80%. The staining intensity was scored as “0” (no staining), “1” (weakly stained), “2” (moderately stained) and “3” (strongly stained). Both percent positivity of cells and staining intensity were decided in a double-blinded manner. The final Tiam1 expression score was calculated with the value of percent positivity score × staining intensity score, which ranged from 0 to 12.35
RNA extraction and real-time PCR
Total RNA from cells was extracted using Trizol reagent (Invitrogen) according to the manufacturer's instructions. DNase-I-treated total RNA (∼1 μg) was used as starting material, to which 1 μl (1 μg/μl) of oligo (dT) primer was added, and DEPC-treated water was added to the total volume of 11 μl. The RNA mixes were heated at 65°C for 5 min and then chilled on ice. To each RNA mix the following components were added: 4 μl of 5× first-strand buffer, 2μl of 0.1 M DTT, 1 μl of 10 mM dNTPs and 1 μl of ribonuclease inhibitor. The samples were incubated at 42°C for 2 min, followed by addition of 200U of Superscript II and incubation at 42°C for 50 min. The reaction was inactivated at 70°C for 5 min. The following primers were used for amplification of Tiam1: sense primer, 5′-TAAGAAGCTGCTGTGCTACG-3′ and antisense primer, 5′-GACTCGTCATACTCCTGCTT-3′. β-actin was amplified as an internal control using sense primer, 5′-AAGACGTACTCAGGCCATGTCC-3′ and antisense primer, 5′-GACCCAAATGTCGCAGTCAG-3′. Real-time PCR was performed using SYBR Green PCR kit (s) following the manufacturer's protocol as previously described.36 The annealing temperature was 56°C for 40 sec. The relative quantification was given by the Ct values, determining the reactions for each target gene and the internal control gene (β-actin) in tumor and normal samples. ΔCt [ΔCt = Ct (target gene) − Ct (β-actin gene)] values were calculated for each cell lines. Relative expression level was determined as 2, where ΔΔCt = ΔCt (HCC cell line) −ΔCt (normal hepatocyte line).
Western blotting analysis
Cells were washed twice with cold phosphate-buffered saline (PBS) and lysed on ice in RIPA buffer (1× PBS, 1% NP40, 0.1% sodium dodecyl sulfate (SDS), 5 mM EDTA, 0.5% sodium deoxycholate and 1 mM sodium orthovanadate) with protease inhibitors. Protein lysates were resolved on 6% SDS polyacrylamide gel, electrotransferred to polyvinylidene fluoride membranes (Immobilon P; Millipore, Bedford, MA) and blocked in 5% nonfat dry milk in Tris-buffered saline, pH 7.5 (100 mM NaCl, 50 mM Tris and 0.1% Tween-20). Membranes were immunoblotted overnight at 4°C with anti-Tiam1 polyclonal antibody (Santa Cruz Biotechnology), anti-β-actin antibody (Santa Cruz Biotechnology), followed by their respective horseradish peroxidase-conjugated secondary antibodies. Signals were detected by enhanced chemiluminescence (Pierce, Rockford, IL).
The correlation of Tiam1 expression to various clinicopathological parameters was evaluated with χ2 test. Overall survival was calculated from the date of surgery to the date of death. Survival analyses were performed according to the Kaplan-Meier method. Cox proportional-hazard analysis was used for univariate and multivariate analysis to explore the effect of variables on survival. The SPSS 13.0 software was used for all statistical analyses and a p value of <0.05 was considered significant.
Expression of Tiam1 in HCC tissues
Tiam1 expression was determined by IHC in the 152 surgical specimens of HCC. Ninety-seven of them (63.8%) exhibited overexpression of Tiam1 (Tiam1 2+ to 3+), and downexpression (Tiam1 0 to 1+) was found in the other 55 cases (36.2%, Table I). As shown in Figure 1, the immunoreactivity of Tiam1 was detected at variable levels and localized in the cellular cytoplasm. The Tiam1 protein expression was negative in normal liver tissues, weak in cirrhotic liver and strong in HCC tissues (Fig. 1). The statistical evaluation of immunohistochemical checking, however, indicated no statistically significant relationship between Tiam1 expression and clinicopathological parameters derived from clinical materials, follow-up data and pathological findings (Table I).
Table I. Relationship Between Tiam1 Expression and Clinicopathologic Features of HCC Patients
Tumor size (cm)
Serum AFP (ng/ml)
Expression analysis of Tiam1 by real-time PCR and Western blot
Real-time PCR analysis and Western blotting analysis were done in the normal hepatocyte line (HL-7702) and HCC cell lines (Hep3B, HepG2, BEL-7402, BEL-7404, BEL-7405, SMMC-7721, QGy-7701, QGy-7703). All the 8 HCC cell lines showed higher level expression of Tiam1 mRNA in comparison with the normal hepatocyte line (Fig. 2a). Further analysis of western blotting revealed that Tiam1 was overexpressed in all the HCC cell lines, whereas it was undetectable in the normal hepatocyte line (Fig. 2b).
Correlation between Tiam1 expression and patients' survival
The prognostic effect of Tiam1 on HCC patients' overall survival was compared between patients with high and low Tiam1 protein levels. By Kaplan-Meier curve assessment, it was observed that high Tiam1 protein level was a significant prognostic factor for poor overall survival in HCC patients. Patients with high Tiam1 protein level had a significantly lower 5-year survival rate than those with low Tiam1 protein level (Fig. 3, p < 0.05). Kaplan-Meier survival curves for low IHC expressions versus high IHC expressions of Tiam1 in the 152 HCC patients also showed a significant separation (p = 0.008, log-rank test).
Univariate and multivariate analyses of prognostic variables in HCC patients
To identify the variables of potential prognostic significance in all the patients with HCC, univariate analysis of each variable was performed in relation to the survival time. The difference in predicting the prognosis was assessed by examining the ratio hazard and p value for each variable. The relative importance of each variable was then determined by multivariate Cox proportional hazards model analysis. From univariate analysis, stepwise inclusion of variables in the model showed that the significant prognostic factors were Tiam1 expression, tumor size, recurrence, metastasis and serum AFP. Multivariate analysis results showed that Tiam1 expression, tumor size and serum AFP might play a role in predicting the overall survival in HCC patients (Table II).
Table II. Univariate and Multivariate Analyses of Individual Parameters for Correlations with Overall Survival Rate: Cox Proportional Hazards Model
HCC is one of the most deadly human carcinomas even with improved diagnosis and compositive therapy, and the prognosis of HCC remains dismal.37 Prognostic molecular biomarkers are valuable for clinicians to predict the risk of disease progression in patients and to aid in tumor control planning. Recently, Engers et al. have found the potential value of Tiam1 as a prognostic molecular biomarker for patients with prostate cancer.
Tiam1, one of GEFs, was originally identified as the invasion- and metastasis-inducing gene by proviral tagging in combination with in vitro selection for invasiveness in T-lymphoma cells. Liu et al. clarified the possible role of Tiam1 gene in the proliferation, invasion and metastasis of CRC and established Tiam1 as a new target for early metastatic diagnostic markers and novel therapeutic strategies.25 Engers et al. found that significantly increased Tiam1 expression levels were not only observed in prostate cancer but also in almost all preneoplastic high-grade prostatic intraepithelium neoplasia lesions, indicating that increased Tiam1 expression occurs early in prostate carcinoma development.35 Interestingly, Engers et al. further concluded that strong Tiam1 overexpression in prostate cancer is an independent prognostic indicator of disease recurrence.
Inspired by the finding of Engers et al., we detected the Tiam1 expression in HCC and explored the clinical prognostic value of Tiam1 by using complete long-term follow-up data of a large cohort of HCC patients and corresponding clinical samples. We have found that the Tiam1 protein is expressed much more strongly in almost all HCCs than in normal liver and cirrhotic liver tissues. We also found that Tiam1 was overexpressed at both transcriptional and translational levels in HCC cell lines. Our results also suggest that Tiam1 might play a role in hepatocarcinogenesis, although the precise molecular mechanism of Tiam1 in HCC tumorigenesis remains to be further elucidated. Our finding, in agreement with many other observations of overexpression of Tiam1 in a variety of cancer tissues, confirms a close association between Tiam1 expression and development and genesis of tumors.
Our contingency table analysis (χ2 test), however, showed that Tiam1 expression does not correlate with any of the clinicopathological parameters analyzed in this study. On the contrary, our Kaplan-Meier survival analysis revealed that high level Tiam1 expression was significantly linked to a poor prognosis of HCC patients after surgical resection (Fig. 3, p = 0.008). This finding may suggest the prognostic value of Tiam1 expression for HCC patients. It is obvious that the expression of Tiam1 in HCC is inversely proportional to the survival time of HCC patients. The higher the expression of Tiam1 gene, the shorter the survival time for HCC patients. By univariate analysis of Cox proportional-hazard model, Tiam1 expression, tumor size, recurrence, metastasis and serum AFP are associated with an increased risk of death from HCC. Moreover, Tiam1 expression, tumor size and serum AFP are singled out as 3 significant and independent prognostic factors relative to overall survival on multivariate analysis. Except Tiam1 expression, the other factors are well-acknowledged indicators of HCC. As Tiam1 expression can be served as a new and independent predictor of decreased disease-free survival for patients with prostate cancer, it may function as a new and independent predictor of prognosis for HCC patients as well.
In summary, our study evaluated the prognostic significance of the Tiam1 expression in a large number of HCC clinical tissue specimens at protein level by immunohistochemical analysis. With great interest, we noticed overexpression of Tiam1 in HCC cell lines as well as in HCC tissues both at transcriptional and translational levels. The most valuable finding of this study is that the overall survival of our study cohort was significantly poorer in high Tiam1 expression cases than in low Tiam1 expression cases. It indicates that Tiam1 expression is a new and independent predictor for HCC patients. Therefore, its clinical value lies in that closer monitoring and more aggressive treatment should be indicated for the HCC patients whose tumor size exceeds 5 cm or whose AFP value exceeds 25 ng/ml or whose Tiam1 protein is overexpressed. This may be a useful way to reduce mortality and prolong the survival time as much as possible. Our finding also provides evidence for molecular target therapy of tumor.
To achieve a better understanding of the mechanism of carcinogenesis and tumor progression in patients with HCC, further molecular, cellular and animal model studies are necessary. Moreover, it will be essential to analyze quantitatively the predictive power of Tiam1 expression for HCC and other tumor types in further studies.