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RARRES3 expression positively correlated to tumour
differentiation in tissues of colorectal adenocarcinoma
R-Y Shyu*
,1
, S-Y Jiang
2
, J-M Chou
3
, Y-L Shih
1
, M-S Lee
4
, J-C Yu
5
, P-C Chao
5
, Y-J Hsu
1
and S-W Jao
5
1
Department of Internal Medicine, Tri-Service General Hospital, 325 Chengung Rd, Sec. 2, Taipei, Taiwan 114, Republic of China;
2
Department of
Microbiology and Immunology, National Defense Medical Center, 161 Minchuan East Rd, Sec. 6, Taipei, Taiwan 114, Republic of China;
3
Department of
Pathology, Tri-Service General Hospital, 325 Chengung Rd, Sec. 2, Taipei, Taiwan 114, Republic of China;
4
School of Public Health, National Defense
Medical Center, 161 Minchuan East Rd, Sec. 6, Taipei, Taiwan 114, Republic of China;
5
Department of Surgery, Tri-Service General Hospital, 325
Chengung Rd, Sec. 2, Taipei, Taiwan 114, Republic of China
RARRES3 is a retinoid-inducible class II tumour-suppressor gene. This study analysed the expression of RARRES3 protein in normal,
adenoma and carcinoma tissues of the colorectum and its correlation with tumour differentiation. The expression of RARRES3
protein in 151 paraffin-embedded colorectal tissues (11 distal normal mucosa, 20 adenoma and 120 colorectal adenocarcinoma) was
determined by immunohistochemistry. RARRES3 protein was expressed in all 11 distal normal, 120 adjacent normal and 20 adenoma
tissues. In distal normal tissues, RARRES3 protein was expressed at the highest levels in differentiated mucosal epithelial cells. Among
120 carcinoma tissues, RARRES3 protein was detected in 97.6% (40 out of 41), 79.4% (54 out of 68) and 17.3% (three out of 11) of
well-, moderately and poorly differentiated tumours, respectively. The expression of RARRES3 protein was positively correlated to
tumour differentiation (test for trend, Po0.0001). Also, levels of RARRES3 protein were found to be higher in the normal tissues
adjacent to 14.6% (six out of 41), 51.5% (35 out of 68), and 90.1% (10 out of 11) of well-, moderately and poorly differentiated
tumours, respectively. The decreases in tumour differentiation and RARRES3 expression were significantly correlated compared to
the adjacent normal tissues (test for trend, Po0.0001). The prognostic implication of RARRES3 protein expression was studied in
107 tumour, and no statistical difference in survival was observed. The expression of RARRES3 protein was positively correlated to
cellular differentiation of normal and adenocarcinoma tissues of the colorectum, which supports the role of RARRES3 in normal and
malignant epithelial differentiation of colorectum. RARRES3 expression was decreased only in carcinoma tissue, which suggests that
altered RARRES3 expression occurs late in colorectal carcinogenesis.
British Journal of Cancer (2003) 89, 146 –151. doi:10.1038/sj.bjc.6601049 www.bjcancer.com
&2003 Cancer Research UK
Keywords: RARRES3; RIG1; TIG3; colorectal carcinoma; differentiation; tumour suppressor gene
Retinoid acid receptor responder 3 (RARRES3), also named as
TIG3 (DiSepio et al, 1998) or RIG1 (Huang et al, 2000), was
isolated from retinoid-treated cells using mRNA differential
display. cDNA of RARRES3 encodes an 18-kDa protein with 164
amino acids. RARRES3 along with HREV107 (Hajnal et al, 1994;
Husmann et al, 1998)and A-C1 (Akiyama et al, 1999) belong to a
family of class II tumour-suppressor genes that block reversible
expression rather than sustained mutation, as a general mechan-
ism of gene inactivation (Sager R, 1997). Proteins of the HREV107
family are shown to suppress transformation induced by H-ras
(Hajnal et al, 1994; Sers et al, 1997; Akiyama et al, 1999) or kinase
activities downstream of the activation of Ras proteins (Huang
et al, 2002). The RARRES3 gene is expressed ubiquitously in
normal tissues, and cancer cell lines have low RARRES3
expression. Ectopic expression of RARRES3 in cancer cells leads
to growth suppression or cellular apoptosis (DiSepio et al, 1998;
Deucher et al, 2000; Huang et al, 2002).
Differential expression of RARRES3 in benign and malignant
tissues has not been extensively studied. RARRES3 expression is
reduced in tissues of basal cell carcinoma and aggressive
squamous cell carcinoma (Duvic et al, 2000). Also, the expression
of RARRES3 is downregulated in B-cell lymphocytic leukaemias
with disease progression (Casanova et al, 2001). Similarly, the
expression of HREV107 is positively correlated to cellular
differentiation. The rat HREV107 protein is expressed in
differentiated epithelial cells of the gastrointestinal tract, and
many cancer cell lines have downregulated HREV107 expression
(Sers et al, 1997). HREV107 is expressed in postmeiotic human
testicular germ cells (Siegrist et al, 2001). Therefore, RARRES3 and
HREV107 may be important in epithelial differentiation, and
altered expression of these proteins may play an important role in
carcinogenesis.
Mechanisms of colorectal tumorigenesis have been studied
extensively. Mutations in the adenomatous polyposis coli tumour-
suppressor gene were proposed to occur early during the
development of polyps, and oncogenic KRAS mutations arose
during the adenomatous stage. The presence of mutations of TP53,
deletions on chromosome 18q (where tumour-suppressor genes
DCC,SMAD2 and SMAD4 have been localised) and mutations of
Received 29 July 2002; revised 20 February 2003; accepted 10 April 2003
*Correspondence: Dr R-Y Shyu, Section of Gastroenterology, Tri-Service
General Hospital, 325 Chengung Rd, Sec. 2, Taipei, Taiwan 114, Republic
of China; E-mail: jsy@ndmctsgh.edu.tw
British Journal of Cancer (2003) 89, 146 – 151
&
2003 Cancer Research UK All rights reserved 0007 – 0920/03
$
25.00
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Molecular and Cellular Pathology
DNA mismatch repair genes coincided with the transition to
malignancy (Vogelstein et al, 1988; Chung, 2000). The RARRES3
gene is localised at chromosome 11q12 (Auer et al, 2002), where
deletion has been observed in tissues of lung and ovary cancer
(Gabra et al, 1995; Iizuka et al, 1995). To investigate the expression
and role of RARRES3 protein in colorectal carcinogenesis, we have
analysed the expression of RARRES3 protein in tissues from the
normal colorectal mucosa, and from adenomas and adenocarci-
nomas using immunohistochemistry (IHC).
MATERIALS AND METHODS
Preparation of RARRES3 antiserum
The RARRES3 peptide corresponding to amino acids 74 –87 of the
RARRES3 protein (Huang et al, 2000) was synthesised and
conjugated to keyhole limpet haemocyanin (Genosys Biotechnol-
ogies Inc, Woodlands, TX, USA). In total, 200 mg of conjugated
peptide antigen mixed thoroughly with Freund’s complete
adjuvant was injected into the popliteal lymph node of New
Zealand white rabbits. The injection was repeated every 2 weeks (a
total of six injections) and consisted of 100-mg RARRES3 peptide
mixed with the incomplete Freund’s adjuvant. Titres of the
antiserum were determined using an enzyme immunoassay. The
specificity of the antiserum was determined by Western blotting of
cytosol extracts prepared from cells expressing the RARRES3-
fusion protein (Huang et al, 2002).
Specimen collection and preparation
A total of 20 adenoma tissues were obtained from 14 male and six
female patients with mean age of 63.3 years. A total of 120 primary
adenocarcinoma with adjacent normal tissues (four from the
caecum, 115 from the colon and one from the rectum) were
obtained from 68 male and 52 female patients with a mean age of
64.2 years. The distribution of tumours according to the level of
differentiation and Dukes’ stages are listed in Table 1. In addition,
11 distal normal tissues were taken from regions 410 cm away
from the bulk of those tumour tissues that had clearly defined
margins. Tissue slides were prepared from paraffin-embedded
blocks with haematoxylin and eosin staining. Each specimen was
evaluated by the same pathologist to define the differentiation
status of carcinoma tissues as well as the degree of dysplasia of
adenoma tissues. The assessment of tumour differentiation is
based on the architectural and glandular differentiation as well as
nuclear features of tumours (Anonymous, 1996). Primary tumours
were staged according to Dukes’ classification system (Dukes,
1932).
Immunohistochemical analysis
Tissue sections were air-dried, deparaffinised, and then boiled
twice for 2 min in 10% DAKO ChemMate
TM
solution (DAKO Co,
Carpinteria, CA, USA) containing 0.05% NP-40 (Nonidet P-40).
The DAKO LSAB
s
2 Peroxidase kit was used to stain the
expression of RARRES3 protein in tissue sections. Tissues were
incubated with RARRES3 antiserum or preimmune serum at a
dilution of 1 to 800 at room temperature for 1 h. The sections were
lightly counterstained with Mayer’s haematoxylin. To verify
antibody specificity, RARRES3 antisera were preincubated with
8–30mg of RARRES3 peptide at 41C overnight. Samples were spun
at 100 000 gat 41C for 10 min before adding the absorbed antisera
to the tissue section. Sections were also stained with p21
WAF1
and
Ki-67 monoclonal antibodies obtained from DAKO Co.
Reviewing and scoring the sections
Patterns of staining, cellular RARRES3 localisation, staining
intensity and percentage of RARRES3 expressed cells were
recorded. The evaluation of staining patterns was performed using
the immunoreactive score (IRS) proposed by Remmele and
Stegner (1987), in which IRS ¼SI (staining intensity) PP
(percentage of positive cells). SI was determined as 0, negative; 1,
weak; 2, moderate; and 3, strong. PP was defined as 1, 0– 9%
positive cells; 2, 10–50% positive cells; and 3, 450% positive cells.
A total of 10 high-power visual fields, with 100 cells per field
counted from different area a of each specimen were chosen at
random for IRS evaluation, and the average of the IRS was
calculated. The final intensity of RARRES3 staining was defined as
‘negative,’ ‘weak’ and ‘strong,’ corresponding to IRS values of 0 – 1,
2–4 and 6 – 9, respectively (Saukkonen et al, 2001). Results of
RARRES3 staining were based on the consensus of the two
investigators (S-Y J, J-M C). Differences in RARRES3 expression
between adjacent normal and carcinoma tissues were based on
final RARRES3 staining.
Statistical analysis
The nonparametric Kruskal–Wallis tests were applied to compare
IRS of RARRES3 associated with tumours at various levels of
Table 1 Expression of RARRES3 protein in colorectal tissues
RARRES3 protein levels (No. (%) of cases)
IRS
a
Tissues No. of cases Negative Weak Strong Mean7s.e.m.
Distal normal 11 0 3 (27.3) 8 (72.7) 5.5571.76
Adenoma 20 7.8071.51
Mild dysplasia 11 0 0 11 (100)
Moderate dysplasia 8 0 0 8 (100)
Severe dysplasia 1 0 0 1 (100)
Tumours
Adjacent normal 120 0 7 (5.8) 113 (94.2) 7.1671.85
Adenocarcinoma 120
Differentiation
Well 41 1 (2.4) 5 (12.2) 35 (85.4) 7.0572.36
a
Moderate 68 14 (20.6) 23 (33.8) 31 (45.6) 4.6873.20
b
Poor 11 8 (72.7) 2 (18.2) 1 (9.1) 1.3671.96
c
Duke’s stages
A+B 51 8 (15.7) 12 (23.5) 31 (60.8) 5.7173.30
C 44 8 (18.2) 11 (25.0) 25 (56.8) 5.2373.16
D 25 7 (28.0) 7 (28.0) 11 (44.0) 4.0473.19
a
IRS ¼immunoreactive score. The values of IRS with different superscripts are significantly different using Kruskal – Wallis test followed by Dunn’s procedure, Po0.05.
RARRES3 expression in colorectal carcinoma
R-Y Shyu et al
147
British Journal of Cancer (2003) 89(1), 146 – 151&2003 Cancer Research UK
Molecular and Cellular Pathology
differentiation as well as Dukes’ stages. Further, Dunn’s procedure
was applied to compare the IRS between groups. Logistic
regression analyses were used to assess the association and trend
between tumour differentiation and chance of positive RARRES3
staining and chance of adjacent normal tissues having RARRES3
expression higher than that of tumours, while controlling for
potential confounding factors, that is, subject’s gender and age.
Survival rates were calculated using the Kaplan –Meier method.
Significance was calculated by the log-rank test. To further validate
the effect of RARRES3 staining on survival, a multivariate Cox
proportional hazard method was used to adjust for age and stage.
RESULTS
Analysis of RARRES3 antisera
The specificity of RARRES3 antiserum was tested on well-
differentiated colon adenocarcinoma tissue using IHC. Tissues
incubated with RARRES3 antiserum showed RARRES3-positive
and granular staining localised at the supranuclear regions of
tumour, adenoma and normal mucosal cells (Figures 1 and 2).
Some random granular patterns of RARRES3 staining were
observed in tumour cells. The staining was specific, since
preincubation of the antiserum with 10 mg of RARRES3 peptide
resulted in the suppression of staining (Figure 1B). No specific
RARRES3 staining was observed in tissues incubated with
preimmune serum (Figure 1C).
RARRES3 expression in normal and adenoma tissues
A total of 11 distal normal tissues and 120 adjacent normal tissues
of carcinomas were analysed for RARRES3 expression by IHC
(Table 1). A total of 72.7 and 94.2% of distal normal and normal
tissues adjacent to carcinomas, respectively, had strong RARRES3
expression with a mean IRS of 5.55 and 7.16, respectively. The
expression of RARRES3, the cell cycle inhibitor p21
WAF1
and
nuclear protein Ki-67 was also analysed in 11 distal normal tissues.
The RARRES3 protein was expressed at the highest levels in
terminal-differentiated mucosal epithelial cells, which expressed
the p21
WAF1
protein (Figures 1D, E). The less-differentiated and
proliferative mucosal crypt cells that were stained positive for the
nuclear protein Ki-67 had decreased or exhibited no RARRES3
expression (Figures 1D, F). RARRES3 expression in 20 adenoma
tissues with dysplasias ranging from mild to severe was analysed.
All 20 adenoma tissues had strong RARRES3 expression regardless
of the variation in the degree of dysplasia (Table 1, Figure 2A).
RARRES3 expression in tissues of colorectal
adenocarcinoma
Levels of RARRES3 protein varied among 120 tissues of colorectal
adenocarcinoma (Table 1). When RARRES3 expression was
analysed with respect to difference in tumour differentiation, 35
out of 41 (85.4%) well-differentiated tissues had strong RARRES3
expression. Among 68 moderately differentiated tumor tissues, 14
tumours (20.6%) did not express RARRES3 protein and 31
tumours (45.6%) had strong RARRES3 expression. Furthermore,
eight out of 11 (72.7%) poorly differentiated colorectal carcinoma
tissues did not express the RARRES3 protein, and two tissues had
weak RARRES3 expression. Representative results of RARRES3
protein expression in well-, moderately and poorly differentiated
tumour tissues are shown in Figure 2. RARRES3 protein levels in
terms of IRS of various differentiations were significantly different
(Po0.0001). The better the tumour tissue differentiation, the
higher the IRS was rated. A significant linear trend was found in
tumour differentiation and RARRES3 expression (Po0.0001). No
significant association was found between RARRES3 IRS and
Dukes’ stages.
We also compared levels of RARRES3 protein between adjacent
normal and tumour tissues within the same tissue slide among 120
carcinoma tissues (Table 2, Figure 2C –H). Levels of RARRES3
expression in 33 out of 41 (80.5%) well-differentiated tissues were
similar to that of the adjacent normal tissues, and six tissues
(14.6%) had RARRES3 expression in normal tissues greater than
that of tumour tissues. The percentage of tissues that showed
higher levels of RARRES3 expression in adjacent normal tissues
was increased to 51 and 90.1% in moderately and poorly
differentiated adenocarcinoma tissues, respectively. Compared to
well-differentiated tumours, moderately and poorly differentiated
carcinoma tissues had a significantly increased chance of having
AB
C
DEFG
Figure 1 Immunohistochemical analysis of the specificity of RARRES3 antiserum and expression of RARRES3, p21
WAF1
and Ki-67 proteins in colon
mucosal tissues. Sections of well-differentiated adenocarcinoma of colon were incubated with RARRES3 antiserum (A), RARRES3 antiserum preincubated
with 10 mg of RARRES3 peptide (B) or preimmune serum (C). Magnification X200. Sections from distal normal mucosal tissues of colon were incubated
with antibodies against RARRES3 (D), p21
WAF1
(E), Ki-67 (F) or preimmune serum (G). Magnification X100. Sections were counterstained with Mayer’s
haematoxylin. Arrows indicate positive staining of RARRES3, p21
WAF1
or Ki-67.
RARRES3 expression in colorectal carcinoma
R-Y Shyu et al
148
British Journal of Cancer (2003) 89(1), 146 – 151 &2003 Cancer Research UK
Molecular and Cellular Pathology
higher RARRES3 protein levels in the adjacent normal tissues than
in tumour tissues (Pfor trend o0.0001).
Prognostic impact of RARRES protein expression
Altogether, 107 patients with colorectal adenocarcinoma were
examined for prognosis related to RARRES3 expression. A total of
23 tumours were stained negative for RARRES3 protein, 25
tumours had weak RARRES3 expression and 59 tumours had
strong RARRES3 expression. Kaplan –Meier survival curves for the
different groups are presented in Figure 3. Based on the 107
patients, no difference in survival was found comparing patients
with negative, weak and strong RARRES3 staining in tumours
(P¼0.883). Similarly, multivariate analysis showed no difference
in survival between patients with negative and weak (P¼0.929) or
between negative and strong (P¼0.292) RARRES3 staining in
tumours.
DISCUSSION
RARRES3 is a retinoid-inducible tumour suppressor. This study
shows that RARRES3 protein is expressed in mucosal tissues of
normal colon and rectum, which is correlated to epithelial
differentiation. The premalignant adenoma tissues expressed the
RARRES3 protein at high levels. Within tumour tissues, the
expression of RARRES3 protein is positively correlated to tumour
differentiation. Compared to the adjacent normal mucosal tissues,
tumours with moderately and poorly differentiated adenocarcino-
ma had significantly reduced RARRES3 expression. However,
survival analysis showed that RARRES3 protein was not a
prognostic marker for patients with colorectal adenocarcinoma.
In tissues of normal colorectal mucosa, RARRES3 protein is
expressed at the highest levels in terminal-differentiated epithelial
cells, which coincides with a high level of expression of the cell
cycle inhibitor p21
WAF1
(Reed, 1997). The proliferative, Ki-67-
positive mucosal crypt cells (Gerdes et al, 1984) show low or no
A
C
E
GH
D
F
B
Figure 2 Expression of RARRES3 protein in adenoma, carcinoma and adjacent normal tissues. Paraffin-embedded sections of adenoma (A,B), well- (C,
D), moderately (E, F) and poorly (G,H) differentiated colorectal carcinoma were assayed for RARRES3 protein expression by IHC using RARRES3
antiserum (A,C–H) or preimmune serum (B). Expression of RARRES3 protein in adenoma (A), carcinoma (C,E, and G) and adjacent normal tissues (D,
F,H) are shown. Sections were counterstained with Mayer’s haematoxylin. Original magnification X100 (A,B) and X200 (C–H). Arrows indicate positive
RARRES3 staining.
RARRES3 expression in colorectal carcinoma
R-Y Shyu et al
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British Journal of Cancer (2003) 89(1), 146 – 151&2003 Cancer Research UK
Molecular and Cellular Pathology
RARRES3 expression. Therefore, RARRES3 is correlated to
differentiation and growth arrest of normal mucosal epithelial cells
of the colorectum. Similarly, the highest RARRES3 expression is
found in normal suprabasal epidermis, hair follicles and sebaceous
gland. Therefore, RARRES3 may be associated with terminal
keratinocyte differentiation and growth arrest occurring in the
suprabasal layers (Duvic et al, 2000). Similar to results of normal
colonic mucosa, the expression of RARRES3 protein is positively
correlated to differentiation of adenocarcinoma of the colorectum.
Moderately and poorly differentiated colorectal tumours had
progressive loss of the RARRES3 expression. In addition, in vitro
studies investigating the transient expression of RARRES3 protein
or fusion proteins also detected the growth suppressive and
proapoptotic activity of these proteins in several cancer cells
(DiSepio et al, 1998; Deucher et al, 2000; Huang et al, 2002).
Therefore, RARRES3 functions as a negative growth regulator. In
addition, it may also be important for normal epithelial differentia-
tion. Similar results were also observed for the HREV107 protein,
the expression of which is limited to differentiated epithelial cells
and the postmeiotic testicular germ cells (Sers et al, 1997; Siegrist
et al, 2001). Currently, a result that shows the active role of
RARRES3 or HREV107 in cellular differentiation has not been
reported. Our preliminary results demonstrated that SC-M1 gastric
cancer cells became enlarged and flatten, similar to retinoic acid-
induced differentiation of SC-M1 cells (Shyu et al, 1995), after
expressing the RARRES3-EGFP fusion protein for 28 days (data not
shown). Further investigation will be required to dissect the active
or passive role of RARRES3 in epithelial differentiation.
Genetic alterations have been studied extensively during steps of
colorectal carcinogenesis. Mutations in the adenomatous polyposis
coli and KRAS oncogenes arose in precancerous lesions. Aberra-
tions in TP53, DCC and SMADs coincide with transition to
malignancy (Vogelstein et al, 1988; Chung et al, 2000). In this
study, RARRES3 protein was expressed at high levels in tissues of
normal colorectal mucosa and the precancerous lesion adenoma.
The decrease in RARRES3 expression was only observed in
carcinoma tissues, which are unclassified by the Dukes’ system,
and also observed in tissues at Dukes’ stages A and B. Therefore,
the decrease or loss of RARRES3 expression may occur in the early
stages of colorectal carcinoma. To our knowledge, deletion at
chromosome 11q12, where RARRES3 is localised, has not been
reported in tissues of colorectal carcinoma. Also, downregulation
instead of mutation of the RARRES3 gene was found during
progression of B-cell lymphocytic leukaemia (Casanova et al,
2001). Similarly, we did not detect any mutation in cDNA of
RARRES3 from 11 cancer cell lines (data not shown). Therefore,
epigenetic alteration may play an important role in the decrease in
RARRES3 expression in colorectal carcinoma tissues. Mutation at
KRAS is frequently observed in adenoma tissues (Vogelstein et al,
1988). Our previous studies showed that RARRES3 negatively
regulated signal pathways of extracellular signal-regulated kinase,
c-Jun N-terminal kinase and p38 mitogen-activated kinase, down-
stream kinases following activation of the Ras protein (Huang et al,
2002). It therefore is likely that high levels of RARRES3 expression
in adenoma tissues as observed in this study may prevent
malignant transformation in cells harbouring mutations of the
Ras family genes through negative regulation of downstream signal
pathways of Ras. Further studies on the molecular mechanism of
RARRES3 will be necessary to resolve the issue.
In conclusion, this and previous studies have demonstrated the
close association of RARRES3 expression in differentiated
epidermis and colorectal epithelium. Progressive loss of RARRES3
expression in tissues of moderately and poorly differentiated
colorectal adenocarcinoma supports the tumour-suppressive role
of RARRES3 in moderately and poorly differentiated colorectal
cancer. However, RARRES3 alone was not a prognostic marker for
colorectal adenocarcinoma. RARRES3 protein is shown to
suppress the downstream signal pathway of Ras. The significance
of the loss of RARRES3 expression along with mutations in Ras
will be investigated in the future.
ACKNOWLEDGEMENTS
We thank Ms Chi-Lin Wu and Pao-Wen Chang for their technical
assistance. This study is supported in part by grants from the
National Science Council (NSC89-2314-B016-069, NSC90-2314-B-
016-061), Department of Defense (DOD-90-66) and Tri-Service
General Hospital, Taipei, Taiwan, Republic of China.
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