1 Up-Regulation of Rac Gtpase Activating Protein 1 Is Significantly

Author Manuscript Published OnlineFirst on August 8, 2011; DOI: 10.1158/1078-0432.CCR-11-0557 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

RACGAP1 and HCC recurrence

Up-regulation of Rac GTPase activating protein 1 is significantly associated with the

early recurrence of human hepatocellular carcinoma

Suk Mei Wang1, London Lucien P.J. Ooi2, and Kam M. Hui1,3

Authors' Affiliations: 1Bek Chai Heah Laboratory of Cancer Genomics, Division of Cellular and Molecular Research, Humphrey Oei Institute of Cancer Research, National Cancer Centre, Singapore; 2Department of Surgical Oncology, National Cancer Centre, Singapore and 3Cancer and Stem Cell Biology Program, Duke-NUS Graduate Medical School, Singapore.

Corresponding Author: Kam M. Hui, Division of Cellular and Molecular Research, National Cancer Centre, 11 Hospital Drive, Singapore 169610; Phone: (65) 6436-8337; Fax: (65) 6226-3843; E-mail: [email protected].

Running title: RACGAP1 and HCC recurrence

Key words: human hepatocellular carcinoma; interactome; oligonucleotide gene arrays; prediction of recurrent HCC disease; RACGAP1.

Abbreviations: RACGAP1, Rac GTPase activating protein 1; HCC, hepatocellular carcinoma; HBV, Hepatitis B virus; HCV, Hepatitis C virus; AFP, alpha-fetoprotein.

Grant support: This work was supported by grants from the National Medical Research Council, Biomedical Research Council of Singapore and The Singapore Millennium Foundation.

Downloaded from clincancerres.aacrjournals.org on September 24, 2021. © 2011 American Association for Cancer Research. Author Manuscript Published OnlineFirst on August 8, 2011; DOI: 10.1158/1078-0432.CCR-11-0557 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

RACGAP1 and HCC recurrence

Translational Relevance

Hepatocellular carcinoma (HCC) is the world’s third commonest cause of cancer-related

deaths. Surgery currently offers the only possibility of long-term survival for these

patients. Unfortunately, recurrences occur in more than two thirds of these patients and

confer a dismal prognosis. In this study, we have systematically presented molecular

evidence and provided clinical corroboration of these data to demonstrate that,

independently from clinical risk factors, aggressive early recurrent HCC tumors have their

Rac GTPase-activating protein 1 (RACGAP1) expression significantly up-regulated. For

the first time, our data provides clinical support for possible drug developments targeting

the various important oncogenic signaling molecules in an interactome clinically relevant

to early HCC recurrence. Our results also suggest the importance of RACGAP1 as a

stratification factor for the design of future comparative therapeutic trials which is

especially important for early recurrent HCC tumors.

RACGAP1 and HCC recurrence

Abstract

Purpose: To assess the significance of Rac GTPase-activating protein 1 (RACGAP1) expression in identifying HBV-positive human hepatocellular carcinoma (HCC) patients who are at high risk for recurrent disease.

Experimental Design: The prognostic significance of RACGAP1 was compared with clinicopathologic parameters available at diagnosis using multivariate and log rank test. RACGAP1 expression and outcome in recurrence was compared between 35 patients with recurrence and 41 patients without recurrence using Kaplan-Meier analysis. RACGAP1 targeted molecules and pathways were identified and characterized by inhibition with siRNA duplexes.

Results: Kaplan-Meier analysis demonstrated that the level of RACGAP1 expression is sufficient to predict the early recurrence of HCC: high RACGAP1 expression correlates with high risk of post-resection recurrent HCC (p<0.0005). Silencing of RACGAP1 in Hep3B and MHCC97-H HCC cells with high endogenous RACGAP1 expression inhibited cell migration and invasion. Using IPA, the target molecules silenced in the RACGAP1 interactome were mostly genes related to the mitotic roles of the polo-like kinases. These included PRC1, AURKB, CDC2, ECT2, KIF23, PAK1 and PPP2R5E. In providing clinical corroboration of these results, when expression of these transcripts were analyzed in an expression database that we have established previously for HBV-positive HCC patients, these genes was mostly up-regulated in patients who exhibited early recurrent disease and hence provided important corroboration of these results.

Conclusions: siRNA silencing RACGAP1 mainly targeted genes in an interactome clinically relevant to early HCC recurrence. Besides being an independent informative prognostic biomarker, RACGAP1 could also be a potential molecular target for designing therapeutic strategies for HCC.

RACGAP1 and HCC recurrence

Introduction

Hepatocellular carcinoma (HCC) is the commonest primary cancer of the liver and

is the world’s third most frequent cause of cancer-related deaths, with more than 660,000

deaths per annum (1-5). The major etiological factors of HCC are hepatitis B virus (HBV)

and hepatitis C virus infection (HCV), and various other non-viral related causes such as

aflatoxins, alcohol intake and other causes of liver cirrhosis including non-alcoholic

steatohepatitis (NASH). The prevalence of HCC in Europe and the US 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

clinicopathological features like 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

RACGAP1 and HCC recurrence

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). While 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 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.

Materials and Methods

Patient samples

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 histopathological data provided to the researchers

were rendered anonymous. Cancerous and some of the corresponding distant

RACGAP1 and HCC recurrence

non-cancerous liver tissues were obtained from patients who underwent surgical resection

as curative treatment for HCC. All tumor tissues were divided into two portions and

immediately snap-frozen in liquid nitrogen. Half of the sample was stored in liquid

nitrogen until use while 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 three months and ultrasound scans of the liver

were performed every six months. Computerized tomography (CT) or magnetic resonance

imaging (MRI) scans of the liver were performed 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 months observation period while 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.

Oligonucleotide Gene chips microarray analysis

Global gene profiling experiments of the clinical samples were performed using the

Human Genome U133 Set (HG-U133A and HG-U133B) from Affymetrix (Affymetrix

Inc., Santa Clara, CA, USA) as previously described (18). Gene profiling analyses

following the inhibition of RACGAP1 expression with siRNA duplexes in the MHCC97-H

RACGAP1 and HCC recurrence

human HCC cell line were performed 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., St.

Louis, MO, USA) to perform statistical analyses. The Affymetrix probeset Id for

RACGAP1 is 222077_s_at.

Immunohistochemistry (IHC)

Sections of 6µm 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 -20oC 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.

Cell cultures

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) (20). Hep3B, HepGG2 and PLC/PRF5 cells were

RACGAP1 and HCC recurrence

obtained from The American Type Culture Collection (ATCC). Cells were maintained in

Dulbecco’s Eagle’s medium (DMEM) supplemented with 10% heat-inactivated fetal

bovine serum (Hyclone).

siRNA treatment

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 100nM each of the two 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 72kDa.

RACGAP1 and HCC recurrence

Migration and invasion assay

Migration assays were performed in 24-well plates Boyden chambers with an 8µm

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 MHCC97H cells were transfected

with RACGAP1 R1 and R2 siRNA sequences for 2d, 4d or 6d. 1.5x105 transfected cells

were seeded and plated in 500µl of 0.1% bovine serum albumin (BSA)-DMEM to the

upper chamber. The lower chamber was filled with 750µl of 10% fetal bovine serum

(FBS)-DMEM as the chemo-attractant. After being cultured for 48h, non-invaded 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 min, permeabilized with 0.2% Triton X-100 and mounted in

Vectashield Mounting Media with DAPI (Vector Laboratories). At least three random

fields per insert were counted and a representative field of each experiment was

photographed. Results are expressed as means ± SE of three independent experiments.

Statistical analysis was performed using t test.

Pull-down assays for Cdc42, Rac1 and Rho

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 13,000g for 10 min and aliquots stored frozen (-80°C) until use. For

Cdc42 and Rac1 pull down assay, 1mg of total protein was incubated overnight at 4oC

RACGAP1 and HCC recurrence

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 three times in lysis buffer and bound proteins eluted in 30µl

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 to the fold ratio of

the corresponding untreated null sample which was normalized to 1.

DNA Fragmentation (TUNEL) assay

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 d2, d4 and d6 following transfection. Harvested cells

were fixed with 1% formaldehyde for 20min at 4oC. The fixed cells were then incubated

with a mixture of terminal deoxynucleotidyl transferase (TdT) and fluorescein-labeled

nucleotide mix for 1h at 37oC and stained with propidium iodide (PI) in the presence of

0.5µg/ml DNase-free RNase.

Statistical analysis

Correlation between clinicopathological features and recurrence was performed

using the STATISTICAL PACKAGE FOR THE SOCIAL SCIENCES (SPSS) for

RACGAP1 and HCC recurrence

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 centre 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).

Results

Over-expression of RACGAP1 correlates with early recurrence of HCC

Expression of RACGAP1 was significantly up-regulated in the HCC biopsies

compared to 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 (Figure 1A). More importantly, RACGAP1

expression was significantly up-regulated in primary HCC biopsies from patients who had

recurrent disease within 2 years (N=35) compared to patients who did not have recurrence

(NR, N=41) (fold change = 2.31, p<0.0001, false discovery rate (FDR) = 0.043%), Fig. 1B.

Consistent with results obtained with the microarray analysis, quantitative real-time PCR

studies demonstrated that samples from patients with high risk of recurrent disease had

RACGAP1 expressions significantly up-regulated compared to samples from patients with

low risk of recurrent disease (Fold change = 2.03, p<0.02), Fig. 1C. Immuno-staining

RACGAP1 and HCC recurrence

experiments using paired R and NR samples further validated the observation that

RACGAP1 was up-regulated 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), Figure 1D. RACGAP1 staining was mainly localized in the nuclei. In

addition, significant difference in RACGAP1 expression could be detected between R and

paired surrounding non-tumorous tissues (p=0.011) while no significant difference in

RACGAP1 expression was observed between NR and paired surrounding non-tumorous

tissues (p=0.149). This observed difference in IHC staining could provide a potential

avenue for application in clinical screening.

RACGAP1 over-expression could serve as an independent prognostic factor for

recurrent HCC

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 performed univariate and multivariate

Cox regression analysis to determine if RACGAP1 over-expression 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 demonstrated that

RACGAP1 and HCC recurrence

RACGAP1 over-expression, 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 performed using Fisher Exact Test, similar to that

reported recently by Li et al (21). Table 2 demonstrates 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

months and 24 months was performed in relationship to their RACGAP1 expression. For

both analyses, it was determined that high RACGAP1 expression gave a significantly

shorter recurrence-free duration compared to low RACGAP1 expression (p=0.00005 and

p=0.000017 respectively), Figures 2A and 2B.

Human HCC cells expressing high levels of RACGAP1 correlated with high

migration rates in vitro

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 while HepG2 and PLC/PRF5 cells expressed negligible amount of intrinsic

RACGAP1 (Figure 3). When the ability of these four cell lines to migrate were studied in

vitro by the Boyden chambers, it was demonstrated that MHCC97-H and Hep3B cells gave

much higher migration rates compared to HepG2 and PLC/PRF5 cells (Figure 3).

RACGAP1 and HCC recurrence

Blocking RACGAP1 expression by siRNA reduced cell migration and invasion

activities in the RACGAP1-positive HCC cell lines Hep3B and MHCC97-H

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 d4, Figure 4A. However, the suppression of

RACGAP1 expression in MHCC97-H cells was not complete at d2 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 for subsequent experiments.

The migration and invasiveness of Hep3B and MHCC97-H cells were tested. At

d4 following transfection with R2, the migratory and invasive ability of both Hep3B and

MHCC97-H cells were significantly reduced, Figures 4B and 4C. Representative images

showing the effect of siRNA treatment on the migration and invasion of MHCC9-H cells in

vitro was shown in Figure 4D.

Additionally, transfection with R2 induced significant DNA fragmentation in

Hep3B and MHCC97-H cells at d4 and d6. At d6, there was a 2.1-fold increase in

R2-transfected Hep3B cells that were stained positively for the TUNEL assay compared to

both null and control cells (p=0.001), Figure 5A. The observed increase was more

significant with the MHCC97-H cells (5.7-fold, Figure 5B). Corroborating with these

observations, Western blot further demonstrated 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, Figure 5C. This is

RACGAP1 and HCC recurrence

likely to be associated with the differentiation of RACGAP1 depleted cells as suggested by

O’Brien et al (22).

RACGAP1 regulates the GTPase activity of Cdc42 and Rac1 but not RhoA

Silencing of endogenous RACGAP1 by R2 in MHCC97-H cells further impaired

the GTPase activity of Cdc42 and Rac1 but inactive towards RhoA as demonstrated by

pull-down assays (Figure 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 to control cells whereas the activity of GTP-RhoA was not

significantly affected (fold ratio = 1.1), Figure 6, suggesting that the GAP domain of

RACGAP1 could strongly stimulate Rac1 and Cdc42 GTPase activity but inactive towards

RhoA.

Functional pathway analysis revealed transcripts that were specifically silenced by

R2 in MHCC97-H cells were differentially up-regulated in HCC patients that had

early recurrent disease

To elucidate the signaling pathways that were significantly altered following the

silencing of RACGAP1 expression by R2, we performed pathway analysis with the

differentially expressed genes identified in MHCC97-H cells at d4 post-R2 transfection

using the 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 signalling, cell cycle G2/M DNA

damage checkpoint regulation, ERK/MAPK signalling and Rac signalling were among the

RACGAP1 and HCC recurrence

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 (Figure 7A). Importantly, when expression of these

transcripts were analysed in a database that we have established previously for

HBV-positive HCC patients, it was demonstrated that all these transcripts were

differentially up-regulated in patients that exhibited early recurrent disease (Figure 7B).

Additionally, ECT2 and PRC1, two downstream genes of the RACGAP1 signaling

pathway, were both significantly up-regulated in early recurrent HCC patients (Figure 7C).

These results strongly support the hypothesis that siRNA against RACGAP1 targeted

genes in an interactome clinically relevant to early HCC recurrence.

Discussion

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 hepatocellular carcinoma (HCC). RACGAP1, insofar as we know, has not been

previously implicated in HCC. Recently, we have demonstrated the feasibility of

performing 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 demonstrated by Kaplan-Meier analysis the potential that high

RACGAP1 expression can be an independent adverse prognosticator for early HCC

RACGAP1 and HCC recurrence

recurrence after curative resection (p<0.0005). The up-regulation 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 pre-invasive 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 the most perturbed canonical pathway identified was the mitotic

roles of polo-like kinase (Figure 6A), the major mechanism involved in cell division (30).

The differentially down-regulated 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 signalling were also interconnected through the ERK

pathway via AURKB (Figure 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 down-regulated transcripts detected on silencing RACGAP1

RACGAP1 and HCC recurrence

expression in MHCC97-H cells were KIF23, PRC1, PPP2RE, PPP2RC and PPP2RB that

interacts directly with RACGAP1 while Cdc42 and Rac were also interconnected through

AURKB (Figure 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 up-regulated 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 up-regulated in

samples of early recurrent HCC patients and its expression was repressed following

R2-mediated silencing of RACGAP1 in MHCC97-H cells (Figure 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.

RACGAP1 and HCC recurrence

Acknowledgment

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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RACGAP1 and HCC recurrence

Table 1. Univariate and multivariate analyses demonstrating that RACGAP1 expression could serve as an independent prognostic factor for recurrent HCC

Univariate Multivariate analysis analysis Variable RR (95% CI) p value RR (95% CI) p value Gender 1.56 (0.61-4.02) 0.358 ns Male (n=61) vs female (n=15) Age 1.07 (0.55-2.08) 0.839 ns >60 yr (n=41) vs ≤60 yr (n=35)

Hepatitis 0.585 ns HBV (n=59) vs non-B/C (n=14) 0.76 (0.33-1.76) 0.525 HCV (n=3) vs non-B/C (n=14) 1.47 (0.31-7.09) 0.632 Encapsulation 0.590 ns Partial (n=19) vs no (n=40) 0.81 (0.36-1.82) 0.604 Complete (n=17) vs no (n=40) 0.63 (0.26-1.57) 0.323 Tumor size 1.62 (0.84-3.16) 0.153 ns >5 cm (n=35) vs ≤5 c, (n=41) AFP 0.328 ns 10-300 ng/ml (n=27) vs ≤10 ng/ml (n=31) 1.66 (0.72-3.78) 0.232 >300 ng/ml (n=21) vs <10 ng/ml (n=31) 1.85 (0.78-4.35) 0.161 Lesion 1.18 (0.46-3.04) 0.732 ns Multiple (n=10) vs single (n-66)

Differentiation 0.626 ns G2 (n=42) vs G1 (n=10) 1.58 (0.47-5.38) 0.460 G3 (n=18) vs G1 (n=10) 2.23 (0.62-8.01) 0.218 G4 (n=5) vs G1 (n-10) 1.61 (0.27-9.65) 0.601 Cirrhosis 1.91 (0.95-3.85) 0.048* 2.55 (1.20-5.40) 0.0148* yes (n=41) vs no (n=35) Vascular Invasion 3.17 (1.62-6.21) 0.011** 4.06 (1.93-8.53) 0.0002*** yes (n=29) vs no (n=47) RACGAP1 3.42 (1.64-7.16) 0.001** 2.71 (1.27-5.74) 0.0096* high (n=39) vs low (n=37) Recurrence is defined as recurrent disease occurred within a 2-year time point. RR, relative risk; CI, confidence interval; *P<0.05; **P<0.005; ***P<0.001; ns=not significant

RACGAP1 and HCC recurrence

Table 2: Correlation between RACGAP1 expression and clinicopathologic features of the 76 HCC patients in this study using Fisher Exact Test

RACGAP RACGAP

High Low P value Gender 0.78 Female 7 8 Male 32 29 Age (years) 1 ≤ 60 18 17 > 60 21 20 Hepatitis (HBV or HCV) 0.24 Positive 34 28 Negative 5 9 Tumor Encapsulation* 0.06 Partial or Complete 14 22 None 23 14 Tumor Size (cm) 0.5 ≤ 5 19 21 > 5 20 16 AFP (ng/ml)** 0.0002 ≤ 20 9 25 > 20 29 12 Lesions 0.19 Multiple 3 7 Single 36 30 Differentiation (staging)** 0.62 G1 – G2 25 27 G3 – G4 13 10 Cirrhosis 0.25 Yes 24 17 No 15 20 Vascular Invasion 0.35 Yes 17 12 No 22 25 Recurrence 0.00004 Yes 28 7 No 11 30 * 3 patients not reported ** 1 patient not reported

RACGAP1 and HCC recurrence

Figure legends

Figure 1. Over-expression of RACGAP1 in primary HCC tumors. (A) Expression of

RACGAP1 in primary HCC tumors compared to NN (histologically normal liver tissues

from patients with colorectal metastases) and ST (histologically normal liver tissues of

HCC patients) using Affymetrix gene chips. (B) Expression of RACGAP1 in tumor

tissues of HCC with recurrent (R) and non-recurrent disease within 2 years. (C)

Expression of RACGAP1 as detected by quantitative real-time PCR analysis. P<0.05 is

statistically significant and FC= fold change. (D) Representative images of RACGAP1

expression in tumor and non-tumorous tissues of NR and R HCC patients following IHC

analysis. Using IHC, cells that stained brown were scored as positive and RACGAP1

staining was mainly localized in the nuclei. Insert in lower left corner shows image

obtained under high magnification.

Figure 2. RACGAP1 over-expression could serve as an independent prognostic factor for

recurrent HCC. (A) Recurrent HCC disease ≤ 6 months. (B) Recurrent HCC disease ≤ 24

months. Kaplan-Meier plots demonstrated high RACGAP1 expression was significantly

associated with early HCC recurrence. The p value was generated using log-rank test

between R and NR.

Figure 3. High levels of RACGAP1 correlated with high migration rates of human HCC

cells in vitro as demonstrated by Northern blot analysis, real-time PCR assays and the

Boyden migration chambers in vitro.

RACGAP1 and HCC recurrence

Figure 4. Silencing of RACGAP1 modulated the cell migration and invasive properties of

Hep3B and MHCC9-H cells with high endogenous RACGAP1 expression. (A) Time

course to study siRNA-mediated silencing of RACGAP1 in Hep3B and MHCC9-H cells

using Western blot. Null= untreated controls; C= RNAi universal negative control-treated

cells; R1 and R2= pool 1 and pool 2 respectively of a total three sequences of siRNA

duplexes designed to knock-down RACGAP1 expression (see materials and methods).

Expression of actin acted as the internal loading control for mRNA. (B) Quantitation of

cell migration in Boyden chambers. Data were obtained from 3 independent experiments.

P<0.05 is statistically significant. (C) Quantitation of cell invasion in vitro. Data were

obtained from 3 independent experiments. P<0.05 is statistically significant. (D)

Representative images showing the effect of siRNA treatment on the migration and

invasion of MHCC9-H cells in vitro. DAPI-stained nuclei in blue 2 days after seeding in

insert chamber were shown.

Figure 5. Silencing of RACGAP1 expression induced apoptosis in Hep3B and MHCC9-H

cells. (A) Quantitation of DNA Fragmentation (TUNEL assay) at d4 following siRNA

treatment on Hep3B cells in vitro. Data were obtained from 3 independent experiments.

P<0.05 is statistically significant. (B) Quantitation of DNA Fragmentation (TUNEL assay)

at d4 following siRNA treatment on MHCC9-H cells in vitro. Data were obtained from 3

independent experiments. P<0.05 is statistically significant. The lower panels showed

representative immunofluorescent images obtained after TUNEL assays. The nuclei of

apoptotic cells were stained with green fluorescence. (C) Western blot showing silencing

of RACGAP1 expression activated caspases 9, 7 and PARP. Null= untreated cells; C=

RACGAP1 and HCC recurrence

RNAi negative control-transfected cells; siR2= RACGAP1 siRNA duplexes-transfected

cells.

Figure 6. Pull-down and immune-blot assays demonstrating that silencing of endogenous

RACGAP1 by R2 in MHCC97-H cells impaired the GTPase activity of Cdc42 and Rac1

but not RhoA. The relative signal of each band in the GTP-bound form of the pull-down

experiments were normalized to the total amount detected in the whole cell lysates and

followed by normalization to the untreated control of the same cell lysate. Null= untreated

cells; C=RNAi negative control-transfected cells; siR2=RACGAP1 siRNA

duplexes-transfected cells.

Figure 7. (A) Ingenuity pathway analysis (IPA) of global gene expression profiling of

siRNA-treated MHCC97-H cells. Interactome showing the interactions between

RACGAP1 and the most significant perturbed canonical pathways detected using IPA

software for pathway analysis. The molecules highlighted in green were down-regulated

following siRNA treatment. (B) Comparison of the interactomes obtained following IPA

analysis of differentially down-regulated genes of siRNA-treated MHCC97-H cells and

differentially up-regulated genes in primary tumor samples of HCC patients with recurrent

disease within 24 months. Molecules highlighted in green and red indicated differentially

decreased and increased expression respectively. (C) Comparison of the signal intensity

for RACGAP1, ECT2 and PRC1 between early recurrent (R) and non-recurrent HCC

patients. FDR (or q-value) of 0.05 implies that 5% of significant tests will result in false

positives.

Up-regulation of Rac GTPase activating protein 1 is significantly associated with the early recurrence of human hepatocellular carcinoma

Suk Mei Wang, London Lucien Ooi and Kam M. Hui

Clin Cancer Res Published OnlineFirst August 8, 2011.

Updated version Access the most recent version of this article at: doi:10.1158/1078-0432.CCR-11-0557

Supplementary Access the most recent supplemental material at: Material http://clincancerres.aacrjournals.org/content/suppl/2015/12/11/1078-0432.CCR-11-0557.DC1

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