Upregulation of Rac GTPase-Activating Protein 1 Is Significantly Associated with the Early Recurrence of Human Hepatocellular Carcinoma Free

Hepatocellular carcinoma (HCC) is the commonest primary cancer of the liver and is the third most frequent cause of cancer-related deaths in the world, with more than 660,000 deaths per annum (1–5). The major etiologic factors of HCC are hepatitis B virus (HBV) and hepatitis C virus infection (HCV), and various other nonviral-related causes such as aflatoxins, alcohol intake, and other causes of liver cirrhosis, including nonalcoholic steatohepatitis. The prevalence of HCC in Europe and the United States is increasing and is currently the leading cause of death in patients with cirrhosis, possibly resulting from the transmission of HCV by intravenous drug abuse and a rising prevalence of obesity and diabetes (6, 7). Surgery currently offers the only possibility of prolonged survival for HCC patients. Unfortunately, recurrence occurs in more than two-thirds of these patients despite initial curative intent and converts the situation to a dismal prognosis (8, 9).

It is presently a challenge to identify patients who are at high risk for early recurrence after undergoing potentially curative treatment for HCC and various surrogate clinicopathologic features such as lymphovascular invasion, capsular invasion, satellite lesions, and tumour numbers are often used with varying reliability. Such high-risk patients could potentially benefit from closer surveillance or receive adjuvant novel interventional measures, if they could be accurately identified. DNA microarrays have been widely applied to the study of human cancer and comprehensive and systematic functional analyses of large number of genetic and epigenetic alterations provide unbiased analytical approaches to decipher the molecular heterogeneity of cancer (10, 11). Through these strategies, distinct subclasses of HCC patients based on their differing gene expression patterns, which were also associated with patient survival, were identified, indicating the presence of distinct molecular subtypes of HCC (12–14). However, the identification of the early recurrence of HCC remains a major challenge and the development of new prognostic markers are urgently needed to identify HCC patients who are at higher risk of having recurrence.

Previous studies by our group and others have successfully shown that specific gene expression signatures can be established from frozen and formalin-fixed cancerous tissues to accurately predict early recurrent disease following curative hepatic surgery (15–19). In this context, we have reported a 57-member gene signature earlier for selecting HCC patients who are at higher risk of having recurrent disease (18). Although a number of options for gene set analysis exist, we have chosen to investigate the prognostic significance of individual members of the gene set using multivariate and log-rank test and observed that Rac GTPase-activating protein 1 (RACGAP1), a member of this 57-member gene signature, gave the best ability to predict early recurrent disease and survival outcome. In this study, we report the identification and molecular characterization of RACGAP1 as a clinically relevant prognostic predictor for recurrent HCC disease.

All tissue samples employed in this study were approved and provided by the Tissue Repository of the National Cancer Centre Singapore and conducted in accordance with the policies of its Ethics Committee. Informed consent was obtained from all participating patients, and all clinical and histopathologic data provided to the researchers were rendered anonymous. Cancerous and some of the corresponding distant noncancerous liver tissues were obtained from patients who underwent surgical resection as curative treatment for HCC. All tumor tissues were divided into 2 portions and immediately snap frozen in liquid nitrogen. Half of the sample was stored in liquid nitrogen until use, whereas the other portion was employed for hematoxylin and eosin staining and evaluated by an independent pathologist. All the cancerous tissues studied were made up of at least 70% of cancer cells.

An early recurrence was defined as a recurrence within 2 years after a curative resection. To assess recurrence, all treated HCC patients were monitored by routine clinical follow-up once every 3 months. The level of serum alpha-fetoprotein (AFP) and liver function tests were determined every 3 months and ultrasound scans of the liver were done every 6 months. Computerized tomography (CT) or MRI scans of the liver were done when the serum levels of AFP showed a rising trend or when the ultrasound results indicated the presence of possible recurrent disease. A total of 76 HCC liver biopsies with 24 histologically normal tissues were collected and studied: Thirty-five samples were from patients who had early recurrent disease over the 24-month observation period, whereas 41 did not have early recurrent disease. In addition, samples of histologically normal liver tissues of 10 colorectal cancer patients who had liver metastases resected were used as reference normal liver tissues.

Global gene profiling experiments of the clinical samples were done using the Human Genome U133 Set (HG-U133A and HG-U133B) from Affymetrix (Affymetrix Inc.) as previously described (18). Gene profiling analyses following the inhibition of RACGAP1 expression with siRNA duplexes in the MHCC97-H human HCC cell line were done with Affymetrix Human Genome U133 Plus 2.0 Arrays. The microarray data have been deposited in the European Bioinformatics Institutes of the European Molecular Biology Laboratory database (http://www.ebi.ac.uk/arrayexpress/) and are accessible through ArrayExpress public database with accession numbers E-MEXP-84 and E-TABM-292. Signal intensities were transformed to log₂ base and imported to Partek Genomics Suite software (Partek Inc.) to conduct statistical analyses. The Affymetrix probeset Id for RACGAP1 is 222077_s_at.

Sections of 6 μm were mounted onto Superfrost Plus microscope glass slides (Thermo Fisher scientific) and stored at −80ºC until use. All frozen sections were fixed with chilled 100% acetone at −20°C for 10 minutes prior to incubation with the mouse RACGAP1 MoAb (M01) clone 1G6 (Abnova) followed by adding dextran carrying anti-mouse IgG conjugated to horseradish peroxidase (Chemicon) and positive staining was developed using the Dako REAL EnVision detection system. Isotype-matched mouse IgG2b (Dako) was used as a negative control. Images of stained sections were imported into Image-Pro Plus, Version 7.0 (Media Cybernetics) for quantifying RACGAP1-stained cells.

The human HCC cell line MHCC97-H was a generous gift from Prof. Tang Zhao-You and Dr. Liu Bin Bin (Liver Cancer Institute and Zhongshan Hospital, Fudan University, Shanghai, PR China; ref. 20). Hep3B, HepGG2, and PLC/PRF5 cells were obtained from the American Type Culture Collection. Cells were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% heat-inactivated FBS (Hyclone).

Two pooled RACGAP1 siRNA sequences, R1 and R2, were employed in this study. The sequences for RACGAP1 siRNA duplexes R1 consisted of a mix of 3 different sequences: duplex 1 minus-CAC ACU GUC UGU CUC AGU UCU UGG C, plus-GCC AAG AAC UGA GAC AGA CAG UGU G; duplex 2 minus-UUU ACU GUG CGG UCA CAG CCA GAG A, plus-UCU CUG GCU GUG ACC GCA CAG UAA A, and duplex 3 minus-UUG CCU UGU CGU CCU AGG UUA GUG G, plus-CCA CUA ACC UAG GAC GAC AAG GCA. R2 also consisted of a mix of 3 sequences: duplex 4 minus-UAU ACA GGC CUG UCU CAG UCA GAC C, plus-GGU CUG ACU GAG ACA GGC CUG UAU A; duplex 5 minus-UUC UGC UGC UUC CAU AAA GGC UCU G, plus-CAG AGC CUU UAU GGA AGC AGC AGA A, and duplex 6 minus-UUG AGA AGC UGA UGU UCA GGA GUG G, plus-CCA CUC CUG AAC AUC AGC UUC UCA A. The MHCC97-H cells were transfected separately with 100 nmol/L each of the 2 pooled RACGAP1 R1 and R2 siRNA sequences or with the stealth RNAi negative control medium GC duplex (Invitrogen) using Lipofectamine 2000 (Invitrogen). Using Western blot and cell lysate of HCC cells, the RACGAP1 MoAb (M01) clone 1G6 gave an expected protein band of 72 kDa.

Migration assays were carried out in 24-well plates Boyden chambers with an 8-μm pore size PET membrane (Falcon). Invasion assays were done in a similar way except that each membrane had a thin layer of GFR Matrigel (Clontech), which served as a reconstituted basement membrane in vitro. Hep3B and MHCC97-H cells were transfected with RACGAP1 R1 and R2 siRNA sequences for 2, 4, or 6 days. A total of 1.5 × 10⁵ transfected cells were seeded and plated in 500 μL of 0.1% bovine serum albumin–DMEM to the upper chamber. The lower chamber was filled with 750 μL of 10% FBS–DMEM as the chemoattractant. After being cultured for 48 hours, noninvaded cells in the inserts were removed by using cotton-tipped swabs. The cells that had invaded to the membrane undersurface were enumerated by microscopy following fixation by 10% formaldehyde for 10 minutes, permeabilized with 0.2% Triton X-100 and mounted in Vectashield Mounting Media with 4′,6-diamidino-2-phenylindole (DAPI; Vector Laboratories). At least 3 random fields per insert were counted and a representative field of each experiment was photographed. Results are expressed as means ± SE of 3 independent experiments. Statistical analysis was done using t test.

The activation of Rho family small GTPases was detected using EZ-Detect Cdc42 Activation Kit, Rac1 Activation Kit (both from Pierce Biotechnology) and Rho Activation Kit (Upstate/Millipore). Cell lysate was prepared with ice-cold lysis buffer provided in the kit supplemented with protease inhibitors (Roche Diagnostics). Lysates were clarified by centrifugation at 13,000 × g for 10 minutes and aliquots stored frozen (−80°C) until use. For Cdc42 and Rac1 pull-down assay, 1 mg of total protein was incubated overnight at 4°C with GST-fusion protein containing p21-binding domain (PBD) of human Pak1 (GST-human Pak1-PBD) that specifically binds active (GTP bound) Cdc42 and Rac1. For Rho pull-down assay, the same amount of protein was incubated with GST-fusion protein containing Rho-binding domain (GST-Rhotekin-RBD). Inactive (GDP bound) Cdc42, Rac1, and Rho were washed 3 times in lysis buffer and bound proteins eluted in 30-μL SDS-PAGE sample buffer. Normalized amounts of lysates were loaded on the gel and assessed for the presence of active small GTPases by Western blot using Cdc42, Rac1, and Rho Ab. Pull-down assay was quantified by calculating the fold ratio of the pull-down active GTPase (GTP-Cdc42, GTP-Rac1, and GTP-Rho A) after normalization to the corresponding total protein. The fold ratio obtained was then compared with the fold ratio of the corresponding untreated null sample which was normalized to 1.

The ApoAlert DNA Fragmentation Assay Kit (Clontech) was employed. Triplicate cultures of Hep3B and MHCC97-H cells were transfected with the RACGAP1 R1 and R2 siRNA sequences and harvested at day 2, 4, and 6 following transfection. Harvested cells were fixed with 1% formaldehyde for 20 minutes at 4°C. The fixed cells were then incubated with a mixture of terminal deoxynucleotidyl transferase and fluorescein-labeled nucleotide mix for 1 hour at 37°C and stained with propidium iodide in the presence of 0.5 μg/mL DNase-free RNase.

Correlation between clinicopathologic features and recurrence was done using the statistical package for the social sciences (SPSS) for Windows (version 15.0). A P value of less than 0.05 was taken as statistically significant.

Tukey Biweight is an M-estimator that has the ability to down-weight data points that are far from the data center in the calculation of the mean. The Tukey Biweight is derived using the following calculations: Assuming ‘x’ is the number of gene expression values to be studied, m = median of x; s = median absolute deviation = median (absolute (x) − m); c = cut-off value determined by software based on s; epsilon = internal constant = 0.0001; w = weight of each data point in x based on Tukey curve; u = (x − m)/((c times s) + epsilon); w = (1 − u2)2 for each data point; Tukey Biweight = sum (w times x)/sum (w).

Expression of RACGAP1 was significantly upregulated in the HCC biopsies compared with available paired adjacent matched histologically normal liver tissues (N = 24) as well as histologically normal liver tissues (N = 10) from patients who underwent surgery for metastatic colorectal cancer in the liver (Fig. 1A). More importantly, RACGAP1 expression was significantly upregulated in primary HCC biopsies from patients who had recurrent disease within 2 years (N = 35) compared with patients who did not have recurrence (NR, N = 41; fold change = 2.31, P < 0.0001, false discovery rate (FDR) = 0.043%), Figure 1B. Consistent with results obtained with the microarray analysis, quantitative real-time PCR studies showed that samples from patients with high risk of recurrent disease had RACGAP1 expressions significantly upregulated compared with samples from patients with low risk of recurrent disease (fold change = 2.03, P < 0.02, Fig. 1C). Immunostaining experiments using paired R and NR samples further validated the observation that RACGAP1 was upregulated in HCC biopsies from patients having early recurrence. The frequency of RACGAP1 positively stained cells was significantly higher in samples from patients having early recurrent disease than in NR samples [median positive signal for recurrent HCC samples = 7% (N = 9); median positive signal for NR biopsies = 0.6% (N = 9); P = 0.014, Fig. 1D]. RACGAP1 staining was mainly localized in the nuclei. In addition, significant difference in RACGAP1 expression could be detected between R and paired surrounding nontumorous tissues (P = 0.011), whereas no significant difference in RACGAP1 expression was observed between NR and paired surrounding nontumorous tissues (P = 0.149). This observed difference in immunohistochemistry (IHC) staining could provide a potential avenue for application in clinical screening.

The Tukey Biweight mean of RACGAP1 expression was 4.7876 for all the HCC biopsies studied. We have arbitrarily considered samples with RACGAP1 expression above 4.78 as high RACGAP1 expressers and samples with RACGAP1 expression less than 4.78 were considered as low expressers. We carried out univariate and multivariate Cox regression analysis to determine whether RACGAP1 overexpression could serve as an independent adverse survival prognostic factor for HCC. In univariate analysis, besides vascular invasion and cirrhosis, RACGAP1 expression was significantly associated with the recurrence of HCC (Table 1). RACGAP1 expression gave the relative risk (RR) of 3.42 and P = 0.001 in its association with early recurrent disease following hepatic resection. Multivariate survival analysis using the Cox's regression model also showed that RACGAP1 overexpression, vascular invasion, and cirrhosis were the only independent statistically significant risk factors for HCC recurrence (Table 1). Multivariate analysis suggested that the risk of developing early recurrent disease was increased 2.7-fold for patients with high RACGAP1 expression.

Additional correlation between RACGAP1 expression and clinicopathologic features in the 76 HCC patients were done using Fisher exact test, similar to that reported recently by Li and colleagues (21). Table 2 shows that high RACGAP1 expression significantly correlates with the early recurrence of HCC (P = 0.00004).

Kaplan–Meier analysis on the HCC patients studied (N = 76) over a period of 6 and 24 months was done in relationship to their RACGAP1 expression. For both analyses, it was determined that high RACGAP1 expression gave a significantly shorter recurrence-free duration compared with low RACGAP1 expression (P = 0.00005 and P = 0.000017, respectively, Fig. 2A and B).

Four human HCC cell lines were employed to study the effect of expression of RACGAP1 and their ability to migrate in vitro. It was determined by Northern blot analysis and real-time PCR assays that MHCC97-H and Hep3B cells expressed high levels of RACGAP1, whereas HepG2 and PLC/PRF5 cells expressed negligible amount of intrinsic RACGAP1 (Fig. 3). When the ability of these 4 cell lines to migrate were studied in vitro by the Boyden chambers, it was shown that MHCC97-H and Hep3B cells gave much higher migration rates compared with HepG2 and PLC/PRF5 cells (Fig. 3).

The HCC cell lines Hep3B and MHCC97-H express high endogenous RACGAP1. The effect of silencing RACGAP1 expression in Hep3B and MHCC97-H cells was studied with the siRNA duplexes (R1 and R2) designed for RACGAP1 (see Materials and Methods). Suppression of endogenous RACGAP1 expression for both Hep3B and MHCC97-H cells was most apparent at day 4 (Fig. 4A). However, the suppression of RACGAP1 expression in MHCC97-H cells was not complete at day 2 with R2. This is likely due to the relatively larger amount of RACGAP1 in the MHCC97-H cells. For subsequent experiments, the siRNA duplex R2 was employed.

The migration and invasiveness of Hep3B and MHCC97-H cells were tested. At day 4 following transfection with R2, the migratory and invasive ability of both Hep3B and MHCC97-H cells were significantly reduced (Fig. 4B and C). Representative images showing the effect of siRNA treatment on the migration and invasion of MHCC97-H cells in vitro was shown in Figure 4D.

In addition, transfection with R2 induced significant DNA fragmentation in Hep3B and MHCC97-H cells at day 4 and 6. At day 6, there was a 2.1-fold increase in R2-transfected Hep3B cells that were stained positively for the terminal deoxynucleotidyl transferase–mediated dUTP nick end labeling (TUNEL) assay compared with both null and control cells (P = 0.001, Fig. 5A). The observed increase was more significant with the MHCC97-H cells (5.7-fold, Fig. 5B). Corroborating with these observations, Western blot further showed that molecules relating to apoptosis, including cleaved caspase 9, cleaved caspase 7, and PARP were all activated in Hep3B and MHCC97-H cells following silencing with the siRNA R2 duplexes (Fig. 5C). This is likely to be associated with the differentiation of RACGAP1 depleted cells as suggested by O'Brien and colleagues (22).

Silencing of endogenous RACGAP1 by R2 in MHCC97-H cells further impaired the GTPase activity of Cdc42 and Rac1 but inactive toward RhoA as shown by pull-down assays (Fig. 6). Reduction in GTP-Cdc42 and GTP-Rac1 activities was prominent in RACGAP1-depleted MHCC97-H cells (fold ratio = 0.43 and 0.4, respectively) when compared with control cells, whereas the activity of GTP-RhoA was not significantly affected (fold ratio = 1.1, Fig. 6), suggesting that the GAP domain of RACGAP1 could strongly stimulate Rac1 and Cdc42 GTPase activity but inactive toward RhoA.

To elucidate the signaling pathways that were significantly altered following the silencing of RACGAP1 expression by R2, we conducted pathway analysis with the differentially expressed genes identified in MHCC97-H cells at day 4 post-R2 transfection using the Ingenuity Pathway Analysis (IPA) software tool (Ingenuity Systems, Inc.). Functional and gene network analysis with differentially expressed genes identified for R2-treated and universal negative control siRNA-treated cells revealed canonical pathways of mitotic roles of polo-like kinase, PI3K/AKT signaling, Wnt/β-catenin signaling, cell-cycle G₂-M DNA damage checkpoint regulation, ERK/MAPK signaling, and Rac signaling were among the most significant canonical pathways altered. Transcripts that were significantly silenced included RACGAP1, PRC1, SFN, CDC2 (CDK1), AURKB, PRSS23, ECT2, SH3RF1, PAK1, TGFB1, KIF23, and PPP2R5E (Fig. 7A). Importantly, when expression of these transcripts were analysed in a database that we have established previously for HBV-positive HCC patients, it was shown that all these transcripts were differentially upregulated in patients that exhibited early recurrent disease (Fig. 7B). In addition, ECT2 and PRC1, two downstream genes of the RACGAP1 signaling pathway, were both significantly upregulated in early recurrent HCC patients (Fig. 7C). These results strongly support the hypothesis that siRNA against RACGAP1 targeted genes in an interactome clinically relevant to early HCC recurrence.

Recurrent HCC disease is the major obstacle in achieving long-term survival outcomes for the treatment of HCC via surgical resections (8, 9). Several studies have addressed the clinical value of gene expression profiling in predicting the early recurrence of human HCC. RACGAP1, insofar as we know, has not been previously implicated in HCC. Recently, we have shown the feasibility of conducting genome-wide expression analysis to derive gene signatures to identify HCC patients who are at higher risk for early recurrence and who could potentially benefit from more intense surveillance and, possibly, adjuvant disease management. RACGAP1 is one of the genes identified in a signature for predicting risk of early recurrence (18). In this study, we further showed, by Kaplan–Meier analysis, the potential that high RACGAP1 expression can be an independent adverse prognosticator for early HCC recurrence after curative resection (P < 0.0005). The upregulation of RACGAP1 and its association with early HCC recurrence were consistent with reports suggesting that RACGAP1 is linked to more frequent aggressive tumor phenotypes of epithelial ovarian cancer (23), invasive cervical cancer (24), and high-grade breast cancer in the transition from preinvasive to invasive disease (25).

In vertebrate cells, RACGAP1 interacts with KIF23 to form the central spindlin complex which plays an essential role in cytokinesis (26, 27). RACGAP1 has been reported to be involved in controlling the initiation of cytokinesis by regulating ECT2, which in turn induces the assembly of the contractile ring and triggers the ingression of the cleavage furrow to complete cytokinesis via interactions with Rac1, Cdc42, and RhoA (26, 28). RACGAP1 together with ANLN, ECT2, AURKB, PRC1, and KIF23 (MKLP1) are cytokinesis-related cluster genes (29). In this study, analysis of the differentially expressed transcripts obtained from R2- and control siRNA-treated MHCC97-H cells with the IPA software tool revealed that the most perturbed canonical pathway identified was the mitotic roles of polo-like kinase (Fig. 6A), the major mechanism involved in cell division (30). The differentially downregulated transcripts obtained by comparing the gene expression profiles of R2- and control siRNA-treated MHCC97-H cells included KIF23, PRC1, PPP2RE, PPP2RC, and PPP2RB that interacts between RACGAP1 and the mitotic roles of polo-like kinase–mediated mitosis pathway. Further interactome mapping suggested that Cdc42, Rac, and actin cytoskeleton signaling were also interconnected through the ERK pathway via AURKB (Fig. 7A).

Although RACGAP1 plays key roles in controlling cell growth and differentiation, the mechanism by which RACGAP1 contributes to HCC recurrence, however, remains unclear. The differentially downregulated transcripts detected on silencing RACGAP1 expression in MHCC97-H cells were KIF23, PRC1, PPP2RE, PPP2RC, and PPP2RB that interacts directly with RACGAP1, whereas Cdc42 and Rac were also interconnected through AURKB (Fig. 7A). Silencing of RACGAP1 expression in Hep3B and MHCC97-H cells with high endogenous RACGAP1 expression and high metastatic potential resulted in reduced cell migration and invasion, reduction of activated Cdc42 and Rac1, and strongly augmented DNA fragmentation that led to cell death in vitro. In this study, we were able to correlate results obtained from siRNA-mediated silencing of gene expression in MHCC97-H cells with gene profiling results of primary HCC clinical samples. Transcripts that were targeted by siRNA against RACGAP1 genes namely KIF23, PRC1, PPP2RE, PPP2RC, PPP2RB, Cdc42, and Rac were all upregulated in primary HCC biopsies from patients with early recurrent disease. RACGAP1 could regulate the activation of Rac1 and Cdc42 to trigger cytoskeletal reorganization and, consequently, influence cell morphology, cell migration, chemotaxis, and the establishment of cell polarity that may lead to tumor metastases (31, 32). In concordance with this hypothesis, expression of PRC1, the main downstream effector of Rac1 and Cdc42 (33, 34), was also found to be upregulated in samples of early recurrent HCC patients, and its expression was repressed following R2-mediated silencing of RACGAP1 in MHCC97-H cells (Fig. 7B). Therefore, it is likely that RACGAP1 could contribute to cancer progression and metastasis through PRC1 to modulate cytoskeletal and transcription pathways that enhance cell motility, proliferation, and survival.

No potential conflicts of interest were disclosed.

We thank the NCC Tissue Repository for providing the tissue specimens for this study and Professors Tang Zhao-You and Liu Bin Bin for providing the MHCC97-H cell line.

This study was supported by grants from the National Medical Research Council of Singapore, Biomedical Research Council of Singapore, and Singapore Millennium Foundation.

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