Yonago Acta medica 2007;50:9–15

Rho-ROCK Expression Predicts the Prognosis in Patientswith T3/T4 Gastric Cancer
Kenjiro Taniguchi, Shunichi Tsujitani, Naruo Tokuyasu, Takuji Naka, Shigeru
Tatebe, Akira Kondo and Masahide Ikeguchi
Division of Surgical Oncology, Department of Surgery, School of Medicine, Tottori University
Faculty of Medicine, Yonago 683-8504 Japan

A small GTPase Rho protein and an effector ROCK have signiﬁcant roles in cancer adhsion, metastasis, invasion, angiogenesis and cell mortality. We investigated the expressions of RhoA protein and ROCK-1 protein in 100 patients with macroscopically T3/T4
gastric cancer immunohistochemically. The expression of RhoA was detected in gastriccancer specimens from 39 patients and that of ROCK-1 in specimens from 30 patients.
The clinicopathological characteristics of 21 tumors with co-expression of RhoA andROCK-1 proteins (Rho/ROCK ON) were compared with those of the 79 remaining tumors (Rho/ROCK OFF). The percentage of lymph node metastasis positive cases in the
Rho/ROCK ON group (81%) was higher than that in the Rho/ROCK OFF group (66%),
but the difference was not signiﬁcant (P = 0.183). However, the prognosis of the 21 patients with Rho/ROCK ON was signiﬁcantly poorer than that of the 79 with Rho/ROCK
OFF (P = 0.006). Our results indicate that the evaluation of the protein expression of
RhoA and ROCK-1 is useful for predicting the prognosis in patients with T3/T4 gastriccancer.
Key words: gastric carcinoma; prognosis; RhoA; ROCK-1

cytoskeletal proteins in response to Rho is important for the ability of tumor cells to metastasize(Chrzanowska-Wodnicka, 1996; Benitah et al.,2004). Activation of Rho results in binding tovarious effector molecules that elicit downstream
responses (Aznar, 2004). The ROCK family,
Rho-associated serine-threonine protein kinase,is one of the effector molecules of Rho. ROCK isthought to participate in the induction of focal adhesions and stress ﬁbers in cultured cells (Itoh etal., 1999). In addition, it has been suggested that
ROCK-1, one of the isomers of ROCK may play
an essential role in the invasion of cancer cells(Kaneko et al., 2002; Yoshinaga, 2003). Rho
protein overexpression has been reported in the

Small GTP-binding proteins are thought to be
involved in signal transduction pathways that
control sets of essential cellular functions suchas reorganization of the actin cytoskeleton, gene
expression, cell cycle progression, and cell-celladhesion (Malliri and Collard, 2003). The small
GTP-binding protein Rho is part of a subfamilyof the Ras superfamily that includes Cdc42, Rac
and Rho proteins. The Rho family has an important role in cancer development and progression,
including cell-cell adhesion, cell motility, transformation, proliferation, invasion, and metastasis(Suwa et al., 1998; Pan et al., 2004; Kamai etal., 2003). Cancer cell migration is central tothe process of metastasis. Rearranging the actin

Abbreviations: MLC, myosin light chain; PBS, phosphate buffered saline; ROCK, Rho-associated coiled-coil domain
kinase9

K. Taniguchi et al.

progression of human cancers, suggesting that the
Rho/ROCK pathway may be a molecular target
for prevention of cancer invasion and metastasis(Sahai, 2003; Somlyo, 2003).
In order to elucidate the malignant potentialof gastric cancer, multiple genetic alterations including activation of oncogenes and inactivationof tumor suppressor genes have been investigatedvigorously. In studies on the expression levelsof RhoA, RhoB, RhoC, Rac1, Rac2, Rac3 andCdc42, increased expressions of RhoA and Rac1
were related to a higher TNM stage and undifferentiated type in gastric cancer (Pan et al., 2004).
Accumulating clinical and experimental data suggest that RhoA working through ROCK protein
kinases contributes to the invasive and metastatic
abilities of gastric cancer. ROCK inhibitors, suchas Y-27632 or WF-536, have been used in somestudies (Takamura et al., 2001; Nakajima et al.,2003a). Although these inhibitors are potent andselective, it remains a possibility that these compounds inhibit additional kinases, such as PRK/
PKN (Davies et al., 2000). In addition, administration of ROCK inhibitors to whole animals may
block tumor cell invasion by enhancing the barrier
function of host cell layers (Wojciak-Stothard etal., 2001; Nakajima et al., 2003b). Furthermore,ROCK inhibitors would be useful antiangiogenic
chemotherapeutic agents in tumors associatedwith elevated Rho/ROCK expression (Croft et al.,2004). However, Rho/ROCK expression in gastric cancer and its relationship to a patient’s prognosis has yet to be clariﬁed.
Primary T3/T4 gastric cancer frequently
metastasizes to the liver and to the peritoneum,and the prognosis of such cases remains unfavorable. In the present study, we immunohistochemically detected the protein expression of RhoA andROCK-1 in patients with T3/T4 gastric cancer
who underwent curative gastrectomy. We also
investigated the clinical signiﬁcance of the Rho/
ROCK pathway in advanced gastric cancer withspecial reference to the mode of recurrence andsurvival of the patients.

Materials and Methods
Patients
Primary T3/T4 gastric adenocarcinoma specimens were obtained from 100 patients who underwent curative gastrectomy at our institution from1976 to 1995. The patients’ ages ranged from 31
to 91 years (average 64.8 years); 64 were male and
36 were female. The clinicopathological ﬁndings
were determined according to the Japanese Classification of Gastric Carcinoma by our hospital
pathologists (Japanese Research Society for Gastric Cancer, 1995). All patients had undergone
either distal, proximal or total gastrectomy with
dissection of the level 1 and 2 regional lymph
nodes (D2). The patients’ follow-up periods forsurvivors ranged from 2 to 216 months (median,
42 months) after surgery. Informed consent was
obtained from all subjects and/or their guardians.
Immunohistochemistry
The methods used for immunostaining for RhoA
protein were as described previously (Horiuchi etal., 2003). A streptoavidin-biotin kit (Histofine
SAB-PO kit; Nichirei, Tokyo, Japan) was used forformalin-ﬁxed and parafﬁn-embedded specimens.
Sections (4 μm in thickness) were dewaxed inxylene, rehydrated in ethanol and then heated in
an autoclave oven (120˚C) for 20 min for retrievalof antigens. Endogenous peroxidase was blockedwith 0.3% hydrogen peroxide in methanol for 20
min. Tissue sections were then washed twicewith phosphate buffered saline (PBS) and preblocked for 60 min with 10% rabbit serum. Afterwashing with PBS, the samples were incubatedovernight at 4˚C with anti-RhoA monoclonal antibody (sc-418, Santa Cruz Biotechnology, Santa
Cruz, CA) at a dilution of 1:100. Sections werethen washed three times in PBS and incubatedwith the secondary anti-mouse immunoglobulin(IgG + IgA + IgM) conjugated with biotin for 60
min, followed by incubation with a streptavidin10

Rho-ROCK expression in gastric cancer

A

B

Fig. 1. Immunohistochemical staining pattern of RhoA and ROCK-1 in gastric cancer. Protein expressions of RhoA
and ROCK-1 were detected in gastric cancer cell nuclei. A: RhoA positive case. B: ROCK-1 positive case. Bar =100 μm (both in A and B).

peroxidase complex for another 60 min. After
three additional washes in PBS, diaminobenzidine
tetrahydrochloride working solution was applied.
Finally, the slides were counterstained with hematoxylin. Staining for ROCK-1 was performed
with an anti-ROCK-1 polyclonal antibody (c-19,
Santa Cruz Biotechnology) at a dilution of 1:100,
using a similar method to that for the RhoA staining. RhoA and ROCK-1 expressions were not detected in the non-neoplastic region of the resected
specimens. Expressions of RhoA and ROCK-1
were evaluated according to the ratio of positive
cells per specimen as described previously (Liuet al., 2004). The staining pattern of RhoA or
ROCK-1 was classiﬁed as follows: negative (negative or equivocal staining or when less than 20%
carcinoma cells were detected) and positive (when
20% and more carcinoma cells were detected).
Two observers who were unaware of the clinicaldata or the disease outcome examined all of thehistological slides. When the interpretation differed between the two observers, a conference
microscope was used for re-evaluation and ﬁnal
decision.

Statistical analysis
The association of factors was evaluated by the
2 -test. The significance of difference amongmeans was determined by the Mann-Whitney U
test. The survival rates were estimated by the
Kaplan-Meier method, and statistical analysis was
carried out by the log-rank test for equality ofthe survival curves. The Cox proportional hazard model and a stepwise procedure assessed theinfluence of each variable on survival. The accepted level of signiﬁcance was P < 0.05.

Results
RhoA/ROCK-I expression and clinicopathological ﬁndings
RhoA and ROCK-1 expressions could not be detected in the non-neoplastic gastric mucosas or in
the underlying stromal cells. They were detected
only in cancer cells (Fig. 1). We decided that 39
cases (39%) were RhoA positive and 30 cases(30%) were ROCK-1 positive (Table 1). RhoA
and ROCK-1 were co-expressed in 21%. No sig11

K. Taniguchi et al.

Table 1. Correlation of RhoA and ROCK-1 expression in 100 gastric cancers

nificant correlation was detected between these
expressions and clinicopathological parameters
including age, sex, tumor size, histology, modeof invasion, lymph node metastasis, lymphaticvascular space invasion and histological stage (datanot shown).
The clinicopathological characteristics of
21 patients with tumors co-expressing RhoA andROCK-1 were compared with those of the other79 patients. The lymph node metastases were observed in 81% of the former group in comparison
to 66% of the latter (P = 0.183, Table 2).

ROCK-1 expression
Positive
Negative
Total
RhoA expression
Positive
Negative
Total

21
9
30

18
52
70

39
61
100

The association of RhoA and ROCK-1 expression: P < 0.001.

mor not expressing both Rho and ROCK-1 diedas well (P = 0.045). Peritoneal metastatic recurrences were frequent in both groups. However, thehematogenic metastases (to liver, lung or bone)
tend to be more frequent in patients with Rho andROCK-1 co-expressing tumors (9/79 [11%], P =0.145, Table 3).

Mode of recurrence
Fifty-two patients died of gastric cancer during
the follow-up period. Fifteen of 21 patients (71%)
with the tumor co-expressing Rho and ROCK-1
died, while 37 of 79 patients (47%) with the tu-

Table 2. Coexpression of RhoA and ROCK-1 proteins and clinicopathological parameters
Both positive (n = 21)

RhoA/ROCK-1 expression
Else (n = 79)P

9.0 ± 5.1

8.7 ± 4.6

0.981

8/13

35/44

0.61

11/10

43/36

0.867

20/1

74/5

0.788

4/17

27/52

0.183

3/18

19/60

0.337

5/16

26/53

0.423

4/16/1

27/44/8

0.233

Size (cm)Histology
Differentiated/Undifferentiated
Mode of invasion
Expansive/Inﬁltrative
Depth of tumor invasion
T3/T4
Lymph node metastasis
Negative/Positive
Lymphatic invasion
Negative/Positive
Vascular invasion
Negative/Positive
Histological stage
II/III/IV

Table 3. Correlation between RhoA/ROCK-1 protein expression and mode of recurrence
Recurrence pattern
Both positive (n = 21)
Peritoneal
Hematogenic
Nodal
Remnant stomach or others
Total

7
5
1
2
15

(33%)
(24%)
( 5%)
( 9%)
(71%)

RhoA/ROCK-1 expression
Else (n = 79)
18
9
4
6
37

12

(23%)
11%)
( 5%)
( 8%)
(47%)

P
0.321
0.145
0.955
0.772
0.045

Rho-ROCK expression in gastric cancer

Prognosis of patients
The overall 5-year survival rate of 100 patients with
T3/T4 gastric cancer was 40%, and the diseasespeciﬁc 5-year survival rate of these patients was
42%. The disease-speciﬁc 5-year survival rate of
21 patients with both RhoA- and ROCK-1-positive
tumors (19%) was signiﬁcantly lower than that of
the remaining 79 patients (48%, P = 0.006, Fig. 2).
Multivariate analysis was performed with
the Cox proportional hazards model using the covariates for histological type, lymph node metastasis, lymphatic invasion, venous invasion and coexpression of RhoA and ROCK-1 (Table 4). The
analysis revealed that lymph node metastasis and
RhoA/ROCK-1 expression independently contributed to shortened survival.

Fig. 2. The disease-speciﬁc survival curves of patientswith T3/T4 gastric cancer according to RhoA/ROCK-1
protein expression. The disease-speciﬁc survival curveof 21 patients with both RhoA- and ROCK-1-positive
tumors (Rho/ROCK ON group) was signiﬁcantly worse
than that of remaining 79 patients (Rho/ROCK OFF
group, P = 0.006).

Discussion

generation and contractility. In ﬁbroblasts grownin tissue culture, RhoA signals through ROCK-1
and ROCK-2 to promote the formation of stressﬁbers and focal adhesions that lead to contractile
actin-myosin bundles (Leung et al., 1996; Uehata
et al., 1997; Riento, 2003). Moreover, genetic
experiments in Drosohila revealed that elevated
Rho signaling in epithelial cells resulted in a lossof epithelial characteristics and increased invasive
migratory behavior (Speck et al., 2003).
Co-overexpression of Rho and ROCK proteins in cancer cells has been reported in ovariancancer, pancreatic cancer, testicular cancer andbladder cancer (Suwa et al., 1998; Kamai et al.,Rho family GTPases are key regulators of manydifferent biological processes including cell motility (Etienne-Manneville and Hall, 2002; Coleman
et al., 2002). When bound to GTP, Rho family
proteins recruit effector proteins that influence
the architecture of the actin cytoskeleton. RhoA
and RhoC activate the serine/threonine kinases
ROCK-1 and ROCK-2. ROCK activation promotes the stabilization of filamentous actin (Factin), phosphorylation of regulatory myosin lightchains (MLCs), the increase in myosin ATPase
activity and the coupling of actin-myosin force

Table 4. Multivariate survival analysis by the Cox proportional hazards model
Variables
Tumor histology
Differentiated/undifferentiatedLymph node metastasis
Positive/negative
Lymphatic invasion
Positive/negative
Vessel invasion
Positive/negative
RhoA/ROCK-1 protein expression
Both positive/else

Number of patients

Hazard ratio

95% Conﬁdential interval

P

43/57

0.669

0.387 – 1.156

0.150

69/31

3.000

1.401 – 6.425

0.005

78/22

0.955

0.452 – 2.019

0.904

69/31

1.180

0.655 – 2.126

0.582

21/79

1.836

1.033 – 3.263

0.039

13

K. Taniguchi et al.

2003, 2004). Among Rho family and ROCK proteins, RhoA and ROCK-1 proteins were reported
to play important roles in carcinogenasis, cancer
cell adhesion, metastasis, invasion and angiogenesis (Itoh et al., 1999; Matsumoto et al., 2001;
Igishi et al., 2003; Horiuchi et al., 2003; Shikada
et al., 2003). On the other hand, RhoB plays atumor-suppressive role in human lung cancer(Mazieres et al., 2004). Thus, to investigate thepossible prognostic value, we analyzed RhoA andROCK-1 protein expressions in advanced gastriccancer immunohistochemically.
In studies on gastric cancer concerning theexpression levels of RhoA, RhoB, RhoC, Rac1,
Rac2, Rac3 and Cdc42, increased expressions of
RhoA and Rac1 were related to a higher TNM
stage and undifferentiated type in gastric carcinoma (Pan et al., 2004). In the current study, 21% of
tumors were positive for both Rho and ROCK-1:
they seemed to have a higher tendency for lymph
node metastasis, lymphatic invasion, and vascularinvasion. They did not show a signiﬁcantly higher
propensity of tumor invasion. However, we foundthat the prognosis of double-positive patients was
signiﬁcantly poorer than that of the other patients.
Also, multivariate survival analysis revealed thatthe double positive tumor was an independent
prognostic factor for advanced gastric cancer aswell as lymph node metastasis. To our knowledge, this is the ﬁrst report to suggest that patientswith RhoA/ROCK-1 double positive gastric cancer have a signiﬁcantly poor prognosis.

4 Coleman ML, Marshall CJ, Olson MF. RAS andRHO GTPases in G1-phase cell-cycle regulation.
Nat Rev Mol Cell Biol 2004;5:355–366.
5 Croft DR, Sahai E, Mavria G, Li S, Tsai J, Lee WM,
et al. Conditional ROCK activation in vivo induces
tumor cell dissemination and angiogenesis. Cancer
Res 2004;64:8994–9001.
6 Davies Sp, Reddy H, Caivano M, Cohen P. Speciﬁcity
and mechanism of action of some commonly used protein kinase inhibitors. Biochem J 2000;351:95–105.
7 Etienne-Manneville S, Hall A. Rho GTPases in cellbiology. Nature 2002;420:629–635.
8 Horiuchi A, Imai T, Wang C, Ohira S, Feng Y,
Nikaido T, et al. Up-regulation of small GTPases,
RhoA and RhoC, is associated with tumor progression in ovarian carcinoma. Lab Invest 2003;83:
861–870.
9 Igishi T, Mikami M, Murakami K, Matsumoto S,
Shigeoka Y, et al. Enhancement of cisplatin-induced
cytotoxicity by ROCK inhibitor through suppressionof focal adhesion kinase independent mechanism
in lung carcinoma cells. Int J oncol 2003;23:1079–
1085.
10 Itoh K, Yoshioka K, Akedo H, Uehata M, Ishizaki T,
Narumiya S. An essential part for rho-associated kinase in the transcellular invasion of tumor cells. Nat
Med 1999;5:221–225.
11 Japanese Research Society for Gastric Cancer. Japanese Classiﬁcation of Gastric Carcinoma. First English Edition. Tokyo: Kanehara; 1995.
12 Kamai T, Tsujii T, Arai K, Takagi K, Asami H, Ito Y,
et al. Signiﬁcant association of Rho/ROCK pathway
with invasion and metastasis of bladder cancer. Clin
Cancer Res 2003;9:2632–2641.
13 Kamai T, Yamanishi T, Shirataki H, Takagi K, Asami H,
Ito Y, et al. Overexpression of RhoA, Rac1, and Cdc42
GTPase is associated with progression in testicular cancer. Clin Cancer Res 2004;10:4799–4805.
14 Ka neko K , Satoh K , Masa mu ne A, Satoh A,
Shimosegawa T. Expression of ROCK-1 in human pancreatic cancer: its down-regulation by morpholino oligo
antisense can reduce the migration of pancreatic cancer
cells in vitro. Pancreas 2002;24:251–257.
15 Leung T, Chen XQ, Manser E, Lim L. The p160 RhoAbinding kinase ROK alpha is a member of a kinase
family and is involved in the reorganization of the cytoskeleton. Mol Cell Biol 1996;16:5313–5327.
16 Liu N, Bi F, Pan Y, Sun L, Xue Y, Shi Y, et al. Reversal of the malignant phenotype of gastric cancer
cells by inhibition of RhoA expression and activity.
Clin Cancer Res 2004;10:6239–6247.
17 Malliri A, Collard JG. Role of Rho-family proteinsin cell adhesion and cancer. Curr Opin Cell Biol
2003;15:583–589.
18 Matsumoto Y, Tanaka K, Harimaya K, Nakatani F,
Matsuda S, Iwamoto Y. Small GTP-binding protein,

References
1 Aznar S, Fernandez-Valeron P, Espina C, Lacal JC.
Rho GTPase: potential candidates for anticancer
therapy. Cancer Lett 2004;206:181–191.
2 Benitah SA, Valeron PF, van Aelst L, Marshall CJ,
Lacal JC. Rho GTPases in human cancer: an unresolved link to upstream and downstream transcriptional regulation. Biochim Biophys Acta 2004;1705:
121–132.
3 Chrzanowska-Wodnicka M, Burridge K. Rho-stimulated contractility drives the formation of stress ﬁbers andfocal adhesions. J Cell Biol 1996;133: 1403–1415.

14

Rho-ROCK expression in gastric cancer

Rho, both increased and decreased cellular motility,
activation of Matrix Metalloproteinase 2 and invasion of human osteosarcoma calls. Jpn J Cancer Res
2001;492:429–438.
19 Mazieres J, Antonia T, Daste G, Muro-Cacho C,
Berchery D, Tillement V, et al. Loss of RhoB expression in human lung cancer progression. Clin Cancer
Res 2004;10:2742–2750.
20 Nakajima M, Hayashi K, Egi Y, Katayama K, Amano
Y, Uehata M, et al. Effect of Wf-536, a novel ROCK
inhibitor, against metastasis of B16 melanoma. Cancer
Chemother Pharmacol 2003a;52:319–324.
21 Nakajima M, Katayama K, Tamechika I, Hayashi K,
Amano Y, Uehata M, et al. WF-536 inhibits metastatic
invasion by enhancing the host cell barrier and inhibiting tumour cell motility. Clin Exp Pharmacol Physiol
2003b;30:457–463.
22 Pan Y, Bi F, Liu N, Xue Y, Yao X, Zheng Y, et al.
Expression of seven main Rho family members ingastric carcinoma. Biochem Biophys Res Commun
2004;315:686–691.
23 Riento K, Ridley AJ. Rocks: multifunctional kinases incell behavior. Nat Rev Mol Cell Biol 2003;4: 446–456.
24 Sahai E, Marshall CJ. Differing modes of tumour
cell invasion have distinct requirements for Rho/
ROCK signaling and extracellular proteolysis. Nat
Cell Biol 2003;5:711–719.
25 Shikada Y, Yoshino I, Okamoto T, Fukuyama S,
Kameyama T, Maehara Y. Higher expression of RhoC
is related to invasiveness in non-small cell lung carcinoma. Clin Cancer Res 2003;9:5282–5286.
26 Somlyo AV, Phelps C, Dipierro C, Eto M, Read P,
Barrett M, et al. Rho kinase and matrix metallopro-teinase inhibitors cooperate to inhibit angiogenesis
and growth of human prostate cancer xenotransplants. FASEB J 2003;17:223–234.
27 Speck O, Hughes SC, Noren NK, Kulikauskas RM,
Fehon RG. Moesin functions antagonistically to the
Rho pathway to maintain epithelial integrity. Nature
2003;421:83–87.
28 Suwa H, Ohshio G, Imamura T, Watanabe G, Arii S,
Imamura M, et al. Overexpression of the rhoC genecorrelates with progression of ductal adenocarcinoma of the pancreas. Br J Cancer 1998;77:147–152.
29 Takamura M, Sakamoto M, Genda T, Ichida T,
Asakura H, Hirohashi S. Inhibition of intrahepatic
metastasis of human hepatocellular carcinoma by
Rho-associated protein kinase inhibitor Y-27632.
Hepatology 2001;33:577–581.
30 Uehata M, Ishizaki T, Satoh H, Ono T, Kawahara T,
Morishita T, et al. Calcium sensitization of smoothmuscle mediated by a Rho-associated protein kinase
in hypertension. Nature 1997:389:990–994.
31 Yoshinaga K, Inoue H, Tana ka F, Mimor i K,
Utsunomiya T, Mori M. Platelet-derived endothelial
cell growth factor mediates Rho-Associated coiled-coli
domain kinase messenger RNA expression and promotes cell motility. Ann Surg Oncol 2003;10: 582–587.
32 Wojciak-Stothard B, Potempa S, Eichholtz T, Ridley
AJ. Rho and Rac but not Cdc42 regulate endothelial
cell permeability. J Cell Sci 2001;114:1343–1355.
Received January 29, 2007; accepted February 13, 2007
Corresponding author: Kenjiro Taniguchi

15