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Tumor Biology |
Department of Thoracic Surgery, Faculty of Medicine, Kyoto University, Sakyo-ku, Kyoto 606-8507 [F. T., Y. O., T. N., Y. K., R. M., M. L., K. Y., H. W.]; Central Clinical Laboratories, Shinshu University Hospital, Matsumoto, Nagano 390-8621 [J. N.]; and Neuron Information Laboratory, National Institute for Physiological Sciences, Myodaiji, Okazaki, Aichi 444-8585 [I. F., K. I.], Japan
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-2-8-linked sialic acid residues and is mainly attached to the
neural cell adhesion molecule (NCAM). Because of the large negative
charge of PSA, presence of PSA attenuates the adhesive property of NCAM
and increases the cellular motility. PSA expression on NCAM is
developmentally regulated, and PSA plays important roles in formation
and remodeling of the neural system through regulation of the adhesive
property of NCAM. Expression of the polysialated form of NCAM has been
also demonstrated in some malignant tumors, such as Wilms’ tumor and
small cell lung cancer. Despite the possible importance as an
oncodevelopmental antigen, however, significance of PSA expression in
most malignant tumors has not been revealed. Therefore, PSA expression
in non-small cell lung cancer was assessed in the present study. PSA
was expressed only in 5 (20.8%) of 24 pathological stage I cases,
whereas it was expressed in most stage IV cases (76.8%, 11 of 14
cases). PSA expression was correlated with nodal metastasis and distant
metastasis, but not with local extent of the primary tumor. Next,
expression of polysialyltransferase genes (PST and
STX genes) which controlled formation of PSA, was
examined. The PST gene was constantly expressed in both
normal lung tissue and tumor tissue of all cases. In contrast, the
STX gene was not expressed in normal lung tissue of any
case, and STX gene expression in tumor tissue was
closely correlated with tumor progression. The STX gene
was expressed only in 1 (4.2%) of 24 stage I cases, whereas it was
expressed in most stage IV cases (85.7%, 12 of 14 cases). These
results suggested that the PSA and STX
genes could be new targets of cancer therapy as well as important
clinical markers. |
INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Carbohydrates expressing on cell surface play important roles in
cell-cell and/or cell-matrix interactions. PSA is a carbohydrate
composed of a linear homopolymer of -2-8-linked sialic acid residues
and is mainly attached to the NCAM (2
, 3)
. Because of the
large negative charge of PSA, presence of PSA attenuates the adhesive
property of NCAM and removal of PSA increases the binding between
NCAM-expressing cells (4)
. It is well known that PSA
expression on NCAM is developmentally regulated; although the
polysialilated form of NCAM (PSA-NCAM) is abundant in a variety of
embryonic tissues, the majority of NCAM in adult tissues lacks PSA. In
the embryonic brain, PSA expressed on NCAM reduces the NCAM adhesion,
which increases cellular motility and allows cell migration and neurite
outgrowth (2
, 3
, 5)
. In the adult brain, although PSA is
lost from NCAM in most areas, PSA-NCAM is expressed only in the
hippocampus and olfactory bulb where structural and synaptic
rearrangement continue even in the adult (2
, 3)
. Thus, PSA
plays important roles in formation and remodeling of the neural system
through regulation of adhesive property of NCAM.
Reexpression of PSA-NCAM has been demonstrated in some malignant tumors, although PSA is lost from NCAM in most adult tissues. Roth et al. (6 , 7) revealed that PSA was expressed and attached to NCAM in Wilms’ tumor. Expression of PSA-NCAM was also found in neuroblastoma (8) , natural killer cell derived-lymphoma (9) , and pancreatic carcinoma with neural invasion (10) , suggesting that PSA-NCAM was closely related to the development of some kinds of malignant tumors. Moreover, expression of PSA-NCAM was found in SCLC, which was characterized by high metastatic potential and rapid cell proliferation (10) . Scheidegger et al. (11) established two sublines, E2 without PSA expression and E3 with PSA expression, from a human SCLC-derived cell line (NCI-H69). Interestingly, the PSA-positive E3 clone demonstrated high metastatic potential, and the PSA-negative E2 clone had very low metastatic potential, although a comparable amount of NCAM was expressed in E3 and E2 clones. The results suggested that PSA allowed PSA-positive cancer cells to detach from primary tumor by attenuating the adhesive property of NCAM, and that PSA was involved with the metastatic potential.
However, there have been few reports on expression of PSA in other malignant tumors, including NSCLC. Although Pujol et al. (12) examined NCAM expression in NSCLC, they did not report on PSA expression. Thus, significance of PSA expression in most malignant tumors has not been revealed despite the possible importance as an oncodevelopmental antigen, probably because tools available for studying on PSA had been limited to antibodies against PSA and endo-N, which specifically recognized and cleavaged PSA (2) . However, two polysialyltransferases that synthesize PSA have been recently cloned, and thereafter studies on PSA have been markedly improved. It has been reported that these two polysialyltransferases, PST [for human (13) , PST-1 for hamster (14) , and ST8SiaII for mouse (15) ] and STX [for human (16, 17, 18) , STX-1 for rat (19) , and ST9SiaIV for mouse (20) ], can independently synthesize PSA. Expression of polysialyltransferase genes is developmentally regulated; both expression of the PST gene and that of the STX gene are strong in the embryonic brain and attenuate in the adult brain like expression of PSA (13 , 16 , 18 , 21) . However, there are some differences between the expression patterns of the PST gene and that of the STX gene. In the adult brain, the PST gene is abundantly expressed in the amygdala, subthalamic nucleus, cerebral cortex, and occipital pole, whereas the STX gene is strongly expressed in the hippocampus, medulla oblongata, and putamen. In other human adult tissues, the PST gene is expressed in a variety of tissues (such as the heart, placenta, spleen, thymus, small intestine, and peripheral blood leukocyte), whereas the STX gene is expressed in limited tissues, such as the heart and thymus (18) . These results suggest that expression of the PST gene and that of the STX gene are independently regulated and that each polysialyltransferase might have different biological roles. However, biological roles of each polysialyltransferase, especially in development and progression of malignant tumors, have not been revealed. In the present study, to clarify the biological and clinical significance of PSA in NSCLC, expression of PSA and that of polysialyltransferase genes in NSCLC were examined.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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IHS.
To detect expression of PSA and NCAM, TSA-Indirect Kit (NEN Life
Science Products, Boston, MA), a sensitive IHS system was used
(24)
. To detect PSA, anti-PSA mAb 12F8 (rat IgM, 500
µg/ml; PharMingen, San Diego, CA), which recognized specifically PSA
(18)
, was used as a primary antibody. To detect a
nonpolysialylated form of NCAM, anti-NCAM (CD56) mAbs 123C3 (mouse
IgG1, 1000 µg/ml; Zymed, South San Francisco, CA) and ERIC-1 (mouse
IgG1, 200 µg/ml; Santa Cruz Biotechnology, Santa Cruz, CA) were used.
All of the procedures were performed following the manufacture’s
protocol. To confirm "true" PSA expression, sections with and
without pretreatment of endo-N were used for every IHS for PSA
(24)
. For pretreatment of endo-N, sections were incubated
with endo-N (50 µg/ml), a kind gift from Dr. Y. Kawase (NGK
Insulators, Ltd., Handa, Aichi, Japan), for 60 min at 37°C (6
, 26
, 27) .
RNA Isolation and RT-PCR.
Total RNA was purified using the RNeasy mini kit (QIAGEN GmbH, Hilden,
Germany), following the manufacturer’s protocol. To digest
contaminated DNA, extracted RNA was incubated with DNase (Nippon Gene,
Toyama, Japan). RT of total RNA was performed using the Ready-To-Go
You-Prime First-Strand Beads and random hexomer (Amersham Pharmacia
Biotech, Piscataway, NJ), following the manufacturer’s protocol. For
accurate evaluation of expression of the STX and
PST genes using RT-PCR assay, cDNA aliquots were diluted at
more than four cDNA concentrations. Measurements were taken in a linear
phase of the reaction where cDNA concentration is directly proportional
to the signal intensity (28)
.
The sense and antisense primers used for PCR amplification of the STX gene were 5'-TCAAGCACAACATCCAGCCAG-3' and 5'-AGGGGTTCATGGTTACCAGGTC-3', which amplified a 399-bp fragment. The sense and antisense primers used for PCR amplification of the PST gene were 5'-ATGTGGAAAGGAGATTGACAG-3' and 5'-AGTGTATACATGAGAAGACCTGT-3', which amplified a 436-bp fragment (20) . PCR primers for the GAPDH gene, used as a internal control, were purchased from Clontech Laboratories Inc. (Palo Alto, CA), and the sequences were 5'-ACCACAGTCCAGCCATCAC-3' and 5'-TCCACCACCCTGTTGCTGTA-3', which amplify a 452-bp fragment. PCR amplification was carried out with an initial 9 min of preincubation at 95°C to activate the AmpliTaq Gold (Perkin-Elmer Corp., Foster City, CA), followed by the following profile: denaturation at 94°C for 30 s, annealing at 60°C for 60 s, and extension at 72°C for 60 s. The number of cycles for STX or PST gene amplification was 35, and that for GAPDH gene amplification was 25. PCR products were electrophoresed on 2% agarose gels, and gels were stained with ethidium bromide. The identities of PCR products were confirmed by DNA sequencing. In every PCR run, cDNA templates obtained from a resected human thymus tissue, in which both STX and PST genes were expressed, were used as a positive control, and tubes containing all ingredients, except for cDNA templates, were also used as a negative control. In addition, in every PCR run, it was confirmed that no PCR product was amplified in tubes containing RNA samples without RT reaction.
Statistical methods.
Counts were compared by the 
2 test. Continuous
data were compared using Student’s t test if the
distribution of samples was normal, or using Mann-Whitney U
test if the sample distribution was asymmetrical. Differences were
considered significant when P was less than 0.05. All
statistical manipulations were performed using the SPSS for Windows
software system (SPSS Inc., Chicago, IL).
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). PSA expression proved to be positive
in 28 (49.1%) of 57 cases with NSCLC (Table 1
and Fig. 1 and E
). The correlation between PSA
expression and the patients’ characteristics was analyzed in NSCLC
cases (Table 2)
and PSA expression proved to be closely correlated with p-stage
(P < 0.002). PSA expression was positive
only in 5 (20.8%) of 24 p-stage I cases, whereas it was positive in 9
(69.2%) of 13 stage III cases and 11 (78.6%) of 14 stage IV cases.
The correlation between PSA expression and each factor of tumor
progression [i.e., extent of primary tumor (T-factor),
nodal involvement (N-factor), or distant metastasis (M-factor)] was
also analyzed (Fig. 2
). Whereas no correlation between PSA expression and T-factor was
demonstrated, PSA expression was significantly correlated with N-factor
and M-factor. PSA was expressed more frequently in node-positive
(N1–3) cases (69.6%, 16 of 23 cases) than in negative (N0) cases
(35.3%, 12 of 34 cases; P = 0.011). PSA was
expressed in most cases with distant metastasis (78.6%, 11 of 14
cases), whereas it was expressed in only 17 of 43 (39.5%) cases
without distant metastasis (P = 0.011).
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Analysis of correlation between PSA expression and tumor
differentiation revealed that PSA was expressed more frequently in
moderately to poorly differentiated tumor than in well-differentiated
tumor (Fig. 3
). Although PSA was expressed more frequently in squamous cell carcinoma
cases than in adenocarcinoma cases, the difference was probably due to
the fact that most adenocarcinomas were classified into
well-differentiated tumor.
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2
3
and Fig. 1F
). Although IHS using ERIC-1, another mAb against
NCAM, demonstrated almost the same staining pattern, the reactivity was
rather weak as compared with that of 123C3. That is, NCAM expression
was detected with ERIC-1 only in six of nine cases that showed positive
NCAM expression with 123C3. In the present study, analysis of NCAM
expression was performed based on results of IHS using 123C3.
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. Western blotting analysis was performed to reveal characteristics of
the carrier molecules of PSA. In some cases showing positive NCAM
expression by IHS, Western blotting analysis revealed a broad band with
a high molecular weight around Mr
200,000 that was digested with endo-N treatment, which seemed to
represent PSA-NCAM (data not shown). In other patients, no apparent
signal was demonstrated with Western blotting analysis, and
characteristics of the carrier molecules of PSA could not be revealed.
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. With
respect to tumor differentiation, NCAM was expressed in only a few
cases with well-differentiated tumor (Fig. 4
).
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and Figs. 5
6
7
). Interestingly enough, the NCAM-negative and PSA-positive groups, not
the NCAM-positive and PSA-positive groups, showed the most advanced
tumor extent. Most cases in the NCAM-negative and PSA-positive groups
had p-stage III or IV disease. In contrast, more than half of the cases
(19 of 29) in the NCAM-negative and PSA-negative groups had p-stage I
disease (Fig. 7
). These results strongly suggested that PSA, not NCAM,
played important roles in tumor progression, especially in nodal and/or
distant metastases.
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). Interestingly, the PST gene was expressed in both normal
lung and tumor tissues of all SCLC and NSCLC cases, whereas
STX was not expressed in normal lung tissue of any case.
Expression of the PST gene in the normal lung was also
confirmed by RT-PCR using normal lung tissues obtained from
noncancerous patients as templates. STX gene expression in
tumor tissues was positive in 25 of 57 (43.9%) NSCLC cases, whereas it
was positive in all SCLC cases (Table 5)
.
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. Moreover,
STX gene expression was significantly correlated with any
factor of tumor progression (T-, N-, or M-factor; Fig. 9
). The STX gene was expressed in most T3–4 cases (84.6%, 11
of 13 cases), in most N1–3 cases (73.9%, 17 of 23 cases), or in most
M1 cases (85.7%, 12 of 14 cases).
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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It has been already revealed using Northern blotting analysis that both expression of the PST gene and that of the STX gene are abundant in the embryonic lung and disappear in the adult lung (18) . However, RT-PCR analysis in the present study revealed that the PST gene was expressed in the adult normal lung tissues, although the STX gene was not. Because the amount of RT-PCR products for the PST gene was smaller, by far, than that for the GAPDH gene, it was suggested that expression of the PST gene could not be detected by Northern blotting. In cancer tissues, the STX gene was expressed only in advanced-stage cases, whereas the PST gene was expressed in all cases, which strongly suggested that the STX gene played critical roles in tumor progression of NSCLC. There have been few reports on the differences between PST and STX (2 , 19 , 30) , and distinct roles of each polysialyltransferase remain unknown. There has been no report on the reason why PST and/or STX are expressed in some adult tissues such as the heart, or no report on the roles of each polysialyltransferase during development of cancer. In the present study, it was proved that expression of PSA in NSCLC was closely correlated with expression of the STX gene. However, it remains unknown whether PSA can be synthesized by STX alone or by STX in cooperation with PST that is constantly expressed in the normal lung.
According to previous reports, -subunit of sodium channels is the
only carrier molecule of PSA other than NCAM in mammalian cells
(31)
; roles of PSA expressed on -subunit of sodium
channels remain unknown. The present study revealed that 19 of 57 NSCLC
cases showed positive PSA expression, although negative NCAM
expression, suggesting that PSA can be attached to molecules other than
NCAM. Recently, Martersteck et al. (32)
have
identified Mr 180,000–260,000
proteins in RBL rat basophilic leukemia cells and MCF7 human breast
cancer cells as possible carrier molecules of PSA, which suggested that
PSA might be more widespread than originally believed. In addition,
Nakayama and Fukuda (33)
reported that PSA could be
attached to fetuin and revealed that NCAM was not required necessarily
in PSA expression. Therefore, PSA can be expressed on some adhesion
molecules other than NCAM, especially during malignant formation, and
PSA may promote formation of metastases by attenuating the adhesive
properties of the "unknown" molecules expressed on NSCLC cells.
In all NCAM-positive cases, PSA expression was also positive regardless of status of STX expression, which suggested that NCAM was a good substrate to which PSA could be attached easily. That is, PSA may be synthesized and attached to NCAM by PST alone, independent of STX. In NCAM-negative cases, PSA expression was almost positive if STX was expressed and was almost negative if STX was not expressed. These results suggested that PSA could not be synthesized and attached easily to carrier molecules other than NCAM when PST alone was expressed. Without NCAM expression, PSA might be synthesized only when STX was expressed with or without PST expression. Although carrier molecules of PSA in NSCLC should be examined in additional studies, the present study clearly demonstrated that PSA itself played critical roles in tumor progression, especially formation of metastatic foci. Thus, PSA and STX can be new targets of cancer therapy as well as important clinical markers in NSCLC.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported by Grant-in-aid 11671317 (to F. T.)
for Scientific Research (C) from the Ministry of Education of Japan. 
2 To whom requests for reprints should be
addressed, at Department of Thoracic Surgery, Faculty of Medicine,
Kyoto University, Shogoin-kawahara-cho 54, Sakyo-ku, Kyoto 606-8507,
Japan. Phone: 81-75-751-3835; Fax: 81-75-751-4647; E-mail: wadah{at}kuhp.kyoto-u.ac.jp
3 The abbreviations used are: NSCLC, non-small
cell lung cancer; PSA, polysialic acid; NCAM, neural cell adhesion
molecule; SCLC, small cell lung cancer; endo-N, endoneuraminidase; IHS,
immunohistochemical staining; RT-PCR, reverse
transcriptation-polymerase chain reaction; GAPDH,
glyceraldehyde-3-phosphate dehydrogenase; mAb, monoclonal antibody;
p-stage, pathologic stage.
Received 10/22/99. Accepted 3/30/00.
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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