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Molecular Oncology, Markers, Clinical Correlates |
Department of Surgical Oncology [K. M., M. K., O. I., T. K., Y. S., M. H., H. O., S. Im.], and Department of Pathology [S. Is.], Osaka Medical Center for Cancer and Cardiovascular Diseases, Higashinari, Osaka 537-8511; Department of Biochemistry [E. M., N. T.], Osaka University Medical School, Suita, Osaka 565-0871; and Diagnostic Research Laboratories, Fuji Rebio Inc. [S. It., H. H., F. T.], Hachioji, Tokyo 192-0031, Japan
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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2
test). Overall 5-year survival rate was 52.8% for GnT-V–positive
patients and 81.7% for GnT-V–negative patients (P < 0.01, Log-rank test). We showed direct evidence for the relationship
between GnT-V and metastasis in human colorectal cancer. Screening of
GnT-V expression in colorectal cancer may provide useful information
for prognosis of postoperative patients. |
INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Modification of cell surface glycoproteins is one of the critical steps
for cellular transformation. In particular, branching of
asparagine-linked oligosaccharides is shown to regulate metastatic
potential in cancer cells (1)
. Among the several patterns
of branching, ß 1–6 branching of
N-acetylglucosamine to 
-D-6
mannoside enhances metastasis in an experimental cancer model of mice
(2)
. This branching is catalyzed by
N-acetylglucosaminyltransferase V
(GnT-V,3
EC 4.1.155), whose
transcription is shown to be regulated by proto-oncogenes, such as the
Ets family (3)
, Src, and erb
B2 (1)
.
The activities of GnT-V in human breast cancer tissues were measured using a synthetic substrate, and increased levels in cancer were reported (4) . mRNA levels of GnT-V were elevated during the hepatocarcinogenesis of rats (5 , 6) , and transforming growth factor ß was shown to stabilize mRNA of GnT-V and increase its activity in mouse melanoma cells (7) . Increased activity of GnT-V in human hepatocellular carcinoma tissues also was reported recently, and positive correlation to tumor size was observed (8) . However, protein expression and localization of GnT-V in human cancer tissues or even in normal tissues have not yet been reported.
Because several biochemical studies suggested that metastatic potential of cancer is in part regulated by GnT-V expression, immunohistochemical study of GnT-V in human cancer tissues should bring about much information regarding to malignant potential. Using human recombinant GnT-V protein as an antigen, we have established a monoclonal antibody against GnT-V and studied protein expression of GnT-V in human colorectal cancer tissues. The comparison of specific GnT-V expression in cancer tissues and conventional clinicopathological analysis raised a possibility of GnT-V expression as a novel, poor prognostic factor for colorectal cancer patients.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Tumor Samples and Patient Follow-up.
Surgical specimens of colorectal cancer patients resected at colorectal
surgery service in the Department of Surgery, Osaka Medical Center for
Cancer and Cardiovascular Diseases were used as histological samples.
Samples were fixed with 20% formalin in PBS for 72 h and embedded
with paraffin. The conventional clinicopathological features and TNM
staging were obtained by the pathologists according to the standard.
The patients were followed up in outpatient clinic of our hospital for
more than 60 months after surgery.
Immunohistochemistry and Pathology Review.
Immunohistochemical analysis of cancer tissue and adjacent noncancerous
mucosa was performed with a monoclonal antibody against GnT-V.
Incubation with the primary antibody was followed by incubation with
biotinylated goat anti-mouse IgG antibody and avidin-horseradish
peroxidase. 3,3'-Diaminobenzidine was used as a chromagen with
hematoxylin counterstain. The sections were examined by two independent
observers without prior knowledge of the clinical status of the
patients.
Statistical Analysis.
Statistical analysis was performed using 2 for
independence test, Fisher’s exact probability test, or Student’s
t test. For survival analysis, the Kaplan-Meier method was
applied, and statistical significance was calculated using the Log-rank
test. Differences were judged statistically significant if P
was <0.01.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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shows a
typical case with positive GnT-V staining in moderately differentiated
adenocarcinoma of colon and negative staining in adjacent noncancer
mucosa. There were 26 cases in which GnT-V was expressed in cancer
tissues, and they were defined as "positive." Among the 26 cases,
one case had GnT-V expression both in cancer and in noncancer mucosa.
The rest of the cases (77 cases) that did not express GnT-V in cancer
tissues were defined as "negative." Among the negative cases, there
was a case with GnT-V expression only in noncancer mucosa (not shown).
The threshold between positive and negative was defined 20% of all
cells observed, but the results were clear-cut, and both of the
observers reached complete agreement with the results of each case.
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. One case with
expression of GnT-V both in cancer and noncancer clearly showed Golgi
localization of GnT-V in noncancer mucosa (Fig. 1D)
.
The Clinicopathological Features of the Patients.
Twenty-six patients had positive staining of GnT-V in cancer tissues,
and clinicopathological features by GnT-V expression were analyzed
(Table 1)
. No association was observed
between GnT-V expression and patients’ gender, age, or tumor size
(maximal diameter). There was also no significant association between
GnT-V and tumor location or histological grade. According to the
standard of TNM classification revised in 1997, lymph vessel invasion
and venous invasion were assessed. No significant relationship was
observed between GnT-V and these invasions.
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. There was not a significant
correlation between GnT-V and T factor (tumor depth) or N factor (lymph
node metastasis). However, GnT-V expression strongly correlated with M
factor (distant metastasis). Of 26 patients with positively stained
GnT-V, 8 patients (31%) had distant metastasis. GnT-V has been shown
to be involved in distant metastasis in an experimental animal model
(2)
, but this is the first report to show the
significant relationship between GnT-V expression and distant
metastasis in cancer patients. Sites of distant metastasis in
GnT-V–positive patients are shown in Table 3
. There was not a significant difference
in terms of sites of metastasis between GnT-V–negative (not shown)
and–positive patients.
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. The site(s) of metastasis, survival
period, outcome, and cause of death in stages II, III, and IV patients
with GnT-V–positive expression are shown in Table 3
. For stage 0 and I
cases, all of the patients including GnT-V–negative cases survived for
more than 5 years. However, for stage II GnT-V–positive patients, 3 of
5 died within 5 years, whereas only 1 of 25 GnT-V–negative patients
died within 5 years (Table 3)
. There was a significant difference in
survival rate between GnT-V–positive and –negative in stage II
(P < 0.01, Log-rank test) (Fig. 3)
. For those cases in stage III, the
survival rate was not significantly different by GnT-V expression. In
stage IV cases, 7 of 8 GnT-V–positive patients died, and 7 of 9
GnT-V–negative patients died within 5 years (not significant). These
data implicate that the poor survival rate in all GnT-V–positive
patients is mainly attributable to the poor survival rate in stage II
patients and partly to the larger number of stage IV patients in
GnT-V–positive cases. Causes of death in GnT-V patients are shown in
Table 3
, and there is no difference between GnT-V–negative (not shown)
and –positive patients.
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. Among the colorectal cancer patients in
stage II, 5 had GnT-V–positive expression, and 25 had negative
expression. Interestingly, the mean value of the size (maximum
diameter) was significantly smaller in GnT-V–positive cases than
negative cases (P < 0.05, Student’s t
test). This implies that GnT-V expression might be a poor prognostic
factor even for small size cancer, which is in general believed
to be less malignant.
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is that GnT-V expression
significantly correlated with venous invasion in stage II cases
(P < 0.05, Fisher’s exact probability test). All of
the patients with GnT-V–positive expression had venous invasion.
Although a larger number is required to make a conclusive statement,
venous invasion in GnT-V–positive patients in stage II could account
for their poor prognosis. |
DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-D-6 mannoside of asparagine-linked
oligosaccharides. The specific activity was measured using a synthetic
substrate (10)
. Although biochemical purification
(11, 12, 13)
and molecular cloning (14
,15)
of
GnT-V were achieved, immunohistochemical study and its prognostic value
have not been reported thus far. This was probably because of the lack
of a specific antibody against GnT-V that facilitates
immunohistochemical study in formalin-fixed, paraffin-embedded
specimens. The monoclonal antibody that we used in this study was
screened for its titer in immunohistochemistry, and the specificity was
confirmed by the competition experiments using an excess amount of
recombinant GnT-V (unpublished data). This antibody prompted us to
first report the expression of GnT-V in human colorectal cancer
tissues. In addition, this report clearly showed the significant
relationship between GnT-V expression and metastasis. As predicted by
the in vitro or animal model study, GnT-V was related to the
malignant potential of the colorectal cancer specimen. Elevated levels of ß 1–6 branching of N-acetylglucosamine, transferred by GnT-V, are shown to be correlated with metastatic potential and tumor invasiveness in several reports. In most cases, phaseolus vulgaris leukoagglutinin binding was used to assess ß 1–6 branching in situ. Dennis demonstrated that increased ß 1–6 branching is not associated with transformation or tumorigenicity but with metastatic potential (2) . An increased level of ß 1–6 branching in human breast and colon neoplasia also was shown using phaseolus vulgaris leukoagglutinin staining that specifically binds to ß 1–6 branching (16) . They reported higher levels of ß 1–6 branching in Dukes C cases, which have lymph node metastasis. Another report showed increase of ß 1–6 branching in human esophageal carcinomas (17) . They showed disposition of ß 1–6 branching predominantly in the outer surface of the tumor adjacent to normal tissues, suggesting that tumor invasiveness is regulated by ß 1–6 branching.
The relationship between GnT-V and malignancy also has been reported. Transcription of GnT-V is induced by src (18) . GnT-V also is shown to be induced by proto-Ha-ras oncogenes in NIH3T3 cells (19) . There are in vitro studies to demonstrate the invasiveness and metastatic potential induced by GnT-V (reviewed in Ref. 1 ). The mobility of Mv1Lu, mink lung epithelial cells, was elevated by the transfection of the GnT-V gene (20) . GnT-V expression causes decreased adhesion to fibronectin and collagen type IV. Transfection of GnT-V into mouse mammary cancer cells resulted in increased lung metastasis (21) . The oncogene her-2/neu induces GnT-V expression and increases its oligosaccharide products (22) . These data supported the positive correlation in our report between metastasis and GnT-V expression in colorectal cancer patients.
The mechanisms underlying this relationship can be speculated by recent advances in glycoprotein biology. Beta 1–6 branching structure, a product of GnT-V, is a good substrate for attachment of poly-N-acetyllactosamine, whose synthesis is controlled by complemental branch specificity of i-extension enzyme and ß 1–4 galactosyltransferase I (23) . This structure is preferentially fucosylated to form sialyl Lewis X, a ligand for selectin in vascular endothelial cells. The level of poly N-acetyllactosamine is increased in highly metastatic colon cancer cells (24) and the level of sialyl Lewis X expression is correlated with poor survival in human colon cancer patients (25 , 26) . From these points, GnT-V may induce tumor metastasis through the formation of sialyl Lewis X on tumor cell surface.
Recently, in pancreatic carcinoma, increased activity of GnT-V is shown (27) . Yao et al. (8) also reported that increased activity of GnT-V was observed in hepatocellular carcinoma. They showed the relationship of GnT-V activity and tumor size assessed by the TNM classification and suggested that GnT-V activity correlated with progression of hepatocellular carcinoma. Our report of colorectal cancer did not show the relationship between tumor size and GnT-V expression. In stage II, we observed even a negative correlation between size (maximal diameter) and GnT-V expression. This is partly because of difference in tumor progression between hepatocellular and colon carcinoma and partly because of the discrepancy between the activity and enzyme expression of GnT-V.
The survival rate of GnT-V–positive patients was significantly less than that of GnT-V–negative patients. This is mainly attributable to the poor prognosis of GnT-V patients in stage II. Colorectal cancer patients in stage II by TNM staging are defined as those negative for lymph node metastasis and free from distant metastasis. Causes of death in stage II are mostly distant metastasis, probably via venous invasion of cancer cells at original sites (28) . The relationship between venous invasion and GnT-V expression and concomitant poor prognosis in stage II patients may provide insights for molecular mechanisms involved in venous invasion. Because overall survival rate is 80% or more in stage II colorectal cancer patients, it is a critical issue whether the adjuvant chemotherapy is necessary or not for those patients (29, 30, 31) . The standard to select patients with poor prognosis in stage II is not currently established. The expression of GnT-V in resected specimen will not only help such selection but also give us information about patients’ prognosis so that intensive follow-up may be done.
Clinical trials are ongoing to test the possibility of swainsonine, an inhibitor for expression of ß 1–6 branched oligosaccharides, as an anticancer drug (32 , 33) . The effects of GnT-V transfection are blocked by swainsonine (20) . Swainsonine inhibits basement membrane invasion of metastatic tumor cells with ß 1–6 branching (34) . Swainsonine (35) also has been shown to reduce tumor metastasis, enhance cellular immune responses, and reduce solid tumor growth in mice. Screening of GnT-V expression in resected cancer tissues may be able to identify postoperative patients eligible for such an inhibitor.
All of the patients in this study were examined for distant metastasis preoperatively and diagnosed for M factor. However, information about GnT-V expression in resected specimens would encourage physicians to detect distant metastasis after surgery because most cases of the distant metastasis after surgery are considered to exist before surgery.
In conclusion, the data we showed raise the possibility of GnT-V expression as a novel prognostic factor for colorectal cancer. GnT-V expression may help physicians to predict metastatic recurrence in stage II, postoperative colorectal cancer patients.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported by grants from Osaka Anti-Cancer
Society, Digestive Molecular Biology (to K. M.), the Second-Term
Comprehensive Ten-year Strategy for Cancer Control from the Ministry of
Health and Welfare (to M. K.); and Grant-in-Aid 10178105 for
Scientific Research on Priority Areas from the Ministry of Education,
Science, Sports and Culture of Japan (to E. M. and N. T.). 
2 To whom requests for reprints should be
addressed, at Department of Surgical Oncology, Osaka Medical Center for
Cancer and CVD, 1-3-3, Nakamichi, Higashinari, Osaka 537-8511, Japan.
Phone: 81-6-6972-1181; Fax:81-6-6981-8055; E-mail: kmurata{at}yo.rim.or.jp
3 The abbreviations used are: GnT-V,
N-acetylglucosaminyltransferase V; TNM, tumor, node,
metastasis.
Received 11/22/99; revised 1/24/00; accepted 1/31/00.
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1–6Manß1–branched asparagine-linked oligosaccharides in murine tissues and human breast carcinomas. Cancer Res., 49: 945-950, 1989.-6-D-mannoside ß-1,6-N-acetylglucosaminyltransferase. J. Biol. Chem., 267: 2920-2927, 1992.-6-D-mannoside ß-1,6-N-acetylglucosaminyltransferase V from a human hepatoma cell line Hep3B. Arch. Biochem. Biophys., 367: 281-288, 1999.[CrossRef][Medline]
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