The expression of mucin genes,. MUCl, 2, 3, 4, 5AC and SB, was determined by northern analyses. Epithelial ovarian cancers expressed several mucins ...
(CANCER RESEARCH 58. 5546-5550.
December I, 1998]
Mucin Gene Expression in Ovarian Cancers1 Robert L. Giuntoli II, Gustavo C. Rodriguez, Regina S. Whitaker, Richard Dodge, and Judith A. Voynow2 De/>anments af Obstetrics and Gynecology ¡R.L. C., G. C. R., K. S. W., R. D.I unti Pediatrics [J. A. V.l. Duke University Medical Center. Durham, North Camlina 277IO
can be detected by antibodies in patients' sera and serve as tumor
ABSTRACT Ovarian cancer ¡sa highly lethal disease with métastasespresent in the majority of patients at the time of diagnosis. The molecular mechanisms underlying the metastatic process of this cancer are not well understood. One family of cell-associated and secreted glycoproteins, the mucin glycoproteins, has been implicated in events leading to metastasis of several epithelial cancers including gastrointestinal and lung cancers. The pur pose of this study was to characterize mucin gene expression in ovarian cancers and relate expression to tumor histology, stage, and patient sur vival. RNA was isolated from 29 epithelial ovarian cancers, 1 neuroendocrine carcinoma, 3 mixed mesoderma! tumors, and two transformed, yet nonmalignant, ovarian epithelial cell lines. The expression of mucin genes, MUCl, 2, 3, 4, 5AC and SB, was determined by northern analyses. Epithelial ovarian cancers expressed several mucins including .\ll'('\. 2,4, and SAC; MUC3 and SB were rarely expressed. In contrast, the trans formed nonmalignant ovarian epithelial cell lines expressed only MUCl and SAC. Although there was no correlation of mucin expression with tumor histology, there was a significant decrease in expression of MUC3 and l/f/4 with increasing cancer stage (/" < 0.05). In addition, a trend toward improved patient survival occurred with increased expression of MUC4. These observations suggest a relationship between mucin gene expression and the metastatic process in epithelial ovarian cancers. Ad ditional investigation of MUCÌ and MUC4 in ovarian cancers may lead to new approaches for early detection and therapy.
INTRODUCTION Ovarian cancer is a highly lethal disease. In the United States, it ranks fourth as a cause of cancer-related deaths among females and accounts for more deaths than all other gynecological cancers com bined ( 1). In the majority of patients, metastatic disease is present at diagnosis. Unfortunately, the long-term survival for women with this disease remains poor. A better understanding of the molecular mech anisms underlying the metastatic process will hopefully lead to novel therapies that can make a dramatic impact on mortality in this disease. In epithelial cancers, the metastatic process has been associated with alterations in cell surface and cell-associated glycoprotein ex pression (2). Specifically, mucin glycoproteins have been implicated in the pathogenesis of epithelial cell malignancies (3-5). Mucin gly coproteins are either membrane-associated and function in cell-cell interactions (3, 6), or they are secreted and constitute the major macromolecular components of mucus in the gastrointestinal, respi ratory, and reproductive tracts (6, 7). Mucin glycoproteins have char acteristic features: (a) their protein backbones (apoproteins) have repetitive domains of peptides rich in serine, threonine, and proline, which are the sites for O-linked glycosylation; and (b) they have multiple, heterogeneous O-linked oligosaccharides that compose up to 50-80% of their mass. Aberrantly glycosylated mucins are overexpressed by tumors and secreted into the circulation of cancer patients. These oligosaccharides Received 6/9/98: accepted 10/5/98. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1This work was supported in part by the American Cancer Society Clinical Oncology Career Development Award 95-85-97 to (G. C. R.) and by NIH Grant R29HL50694 (to J. A. V). • To whom requests for reprints should be addressed, at Pediatrie Pulmonary Diseases. Box 2994. Duke University Medical Center. Durham. NC 27710.
markers (8). Ovarian cancers have several oligosaccharide tumor markers including cancer antigen 125, cancer antigen 19-9, carcinoembryonic antigen, Lewis y antigen (Fucal^>2Galßl —» 4[Fucal^3]GlcNAcßl-*3Galßl^R), and Tn antigen (GalNAcal-» O-Ser/Thr; Rets. 9-12). Because of altered glycosylation, mucin apopro teins (detected by anticancer-associated serum antigen and antimammary serum antigen antibodies) are more readily detected in the sera of both ovarian and breast cancer patients than in normal subjects (13). The etiology of altered mucin glycosylation in cancer cells is not understood; changes in mucin glycosylation may be due to alterations in glycosyltransferase activities (14) or to changes in the specific mucin apoproteins that are produced and then glycosylated in the cancer cells. Recently, by molecular technology, at least 10 different mucin genes have been identified and designated MUC\ to MUC9 (7, 15). By using the cDNA sequences reported for these genes, several studies have demonstrated that alterations in mucin gene expression are associated with the metastatic process in epithelial cell cancers. MUC4 mRNA and glycoproteins are present in high levels in pancre atic cancer tissues but not in benign pancreatic tissue (16, 17). MUC2 mRNA and protein are present in noninvasive pancreatic tumors but not in invasive cancer (18). MÕ/C5ACand MUC5B mRNA expression correlate with earlier postoperative recurrence of lung cancer (19). The presence of MUC5AC glycoprotein also correlates with invasive gastric cancer (20) and pancreatic adenocarcinoma (16). These reports support the concept that changes in mucin expression are related to the metastatic process. Several ovarian epithelial cancers make mucin glycoproteins by histological analysis (21, 22), yet only the expression of MUC2 mRNA (23) and MUCl, 2, and 3 glycoproteins have been character ized in ovarian cancers (24, 25). This report describes the expression of six mucin genes found in ovarian cancer tissues and compares this expression to transformed nonmalignant ovarian cells. Mucin expres sion from ovarian cancer tissues was also related to tumor histology, stage, and patient survival.
MATERIALS
AND METHODS
Cancer Tissues and Cell Cultures.
Thirty-one ovarian cancer tissue sam
ples were obtained at the time of primary surgery and two additional specimens were acquired during surgery for tumor recurrence. All of the tissues were stored at -70°C in the Duke University Medical Center Gynecological On cology tissue bank. A chart review was performed to record the tumor pathology, metastatic stage, therapies, and clinical outcome for each patient. Some patients received either radiation and/or chemotherapy in addition to surgery. No patients received endocrinological. immunological, or other adju vant therapies. These data are reviewed in Table 1. Twenty-nine of the specimens were taken from patients with epithelial ovarian cancer, including 22 serous cystadenocarcinomas. 2 mucinous cystadenocarcinomas, I endometrioid adenocarcinoma. 1 clear cell adenocarcinoma. 1 primary peritoneal adenocarcinoma. and 2 poorly differentiated carcinomas. In addition, tumor samples were obtained from one patient with an ovarian neuroendocrine carcinoma and three patients with mixed mesodermal tumors of the ovary. Two spontaneously transformed, yet nonmalignant. ovarian epithelial cell cultures derived from normal ovarian epithelium were also evaluated for mucin gene expression. These cultures had been established as described previously (26. 27). The ovarian surface epithelial cells were maintained in monolayer culture ina 1:1 mixture of MCDB105 and M199 media (Sigma Chemical Co.,
5546
MUCIN (il-.NI: EXPRESSION
IN OVARIAN
EPITHELIAL
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labeled cDNA at 62°Cfor MUC\. 2, 5AC. and yactin. and at 58°Cfor MUtt,
Table 1 Ovarian cancer histology and stage and patient clinical profiles
4. and 5B. The blots were then washed twice tor 30 min with 0.03 M sodium Sample123456789101112131415161718192021222324252627282930313233Stage1"1A citrate, 0.3 M sodium chloride, and 0.1% SDS at room temperature and then cell''MucinousSerousSerousEndometrioidSerousSerousSerousSerousSerousMMT once for 15 min with 1.5 m.Msodium citrate, 15 mM sodium chloride, and 0.1 % LMPIA SDS at the same temperature as hybridization. The filters were then exposed LMPIBIIC1IC1IBIIBIIC on Kodak X-Omat film at — 70°Cfor I to 4 days for autoradiography. Statistical Analysis. To determine mucin expression from northern autoradiographs for the six MUC genes, two observers—blinded to the identifica tion of the samples—evaluated whether there was a positive signal or no signal LMPIIC LMPn
for each sample. Mucin gene expression was stratified by histological cell type and tumor stage. The x2 test for trend was used to determine whether there
icmemeII
ovaryMMT ovaryMMT ovaryMucinousNeuroendocrinePrim, LMPmemememeIM
were significant differences in the frequency of MUC expression between groups. Differences were considered significant if P < 0.05. Kaplan Meier analysis was used to determine a correlation between mucin gene expression and patient survival.
peritonealSerousPoorly diff.SerousSerousSerousSerousSerousSerousSerousSerousPoorly
RESULTS \mememememememeIVIVIVIVIVRECRECHistologyClear Mucin Gene Expression in Ovarian Cancer Tissues. Northern analyses for MUC\. 2, 3, 4, 5AC, and 5B were performed for ovarian cancer tissues. Most of the MUC mRNA transcripts had a polydis-
perse pattern as demonstrated by the Northern analysis for MUC4 (Fig. IA). Polydisperse mucin transcripts probably represent multiple diff.SerousSerousSerousSerousSerousSerousRadiationNNNYYYNNNNNNNYNNNNKNNNNNNNNNYYNNNNChemotherapyNNNNYYYYNNYYYYNYYNKYYYYYYYYYYYYYYYSu transcript sizes expressed by the tissues: this pattern has been reported previously in respiratory epithelial cells (32) and reproductive tract epithelium (33). In contrast to MUC4 mRNA, MUCl expression was usually present as either one or two discrete bands with the most common transcript size of 4.4 kb as reported previously in respiratory " Tumor stages are described in Ref. l. tissues (Ref. 34; Fig. Iß).The polydisperse pattern was not due to ' Clear cell, clear cell adenocarcinoma; Mucinous. mucinous cysladenocarcinoma: Serous, serous cystadenocarcinoma: Endometrioid. endometrioid adenocarcinoma-. MMT mRNA degradation inasmuch as the ethidium stain of the Northern ovary, mixed mesodermal tumor of the ovary: Prim, peritoneal, primary peritoneal tumor: agarose gels showed that ribosomal bands were intact (Fig. ID) and Poorly diff.. poorly differentiated tumor: Tumors LMP. tumors, with low malignant the Northern for -yactin revealed intact mRNA bands for each sample potential by histologie grade: In one patient, adjuvant NK. adjuvant therapies were not known: N. no: Y, yes: NED. no evident disease; AWD. alive with disease: DOD. death due to disease; LTF. lost to follow-up.
St. Louis. MO) supplemented
with \(Wc heat-inactivated
fetal bovine serum
(Life Technologies. Gaithersburg. MD). The morphology of these cells in culture was consistent with epithelium as verified by immunohistochemistry with anticytokeratin antibodies (27). Two control cell lines/tissues were used: (a) CFPAC' (28). a generous gift from Dr. Ray Fri/.zell (University of Pittsburgh. Pittsburgh, PA), was cultured in Iscove's modified Dulbecco's medium supplemented with 10% fetal bovine serum and penicillin/streptomy cin: and (hi normal human skeletal muscle was obtained from the Department of Pathology. Duke University Medical Center (Durham. NO. cDNA and Oligonucleotide Probes. Probes for MUC\, 2. 5AC and -yactin were synthesized from cDNA clones as was described previously (29). Inserts from these clones were labeled with [ '2P]dCTP using a random primer labeling kit (Prime-It II. Stratagene, La Jolla, CA) to a specific activity of 20 X IO8 cpm/fj.g cDNA. Synthetic 48-bp oligonucleotide
probes for MUC3, 4, and 5B
complementary to the tandem repeat domains (30) were synthesized and labeled to a specific activity of 20 x 10s cpm/fig with ['2P|dATP using T4 polynucleotide kinase (Promega. Madison, WI). Northern Analysis. Total cellular RNA was isolated from the tissue sam ples and cell cultures by the single-step guanidine isothiocyanate-phenolchloroform method using TRIzol reagent (Life Technologies: Ref. 31). RNA was quantitated by spectrophotometry. Northern analysis was performed as described previously (29). Ten /xg of total RNA from each sample were electrophoresed on 1.2% agarose-formaldehyde gel and transferred to nylon membrane (Nytran. Schleicher and Schuell. Keene. NH) by capillary blotting in l M ammonium acetate. Before transfer, the gels were stained with ethidium bromide to evaluate for RNA integrity and loading. After UV cross-linking (Stratalinker, Stratagene, La Jolla, CA). the membranes were prehybridi/.ed in 0.5 M sodium phosphate buffer and hybridi/ed for at least 24 h with ['2P11The abbreviations
used are: CFPAC. pancreatic adenocarcinoma
cystic fibrosis patient: REC, recurrent tumor.
at the appropriate size (Fig. 1C). When the mucin expression was stratified by tumor histology, the serous, mucinous, and endometrioid tumors expressed tour to five of six genes with all of the three histological types expressing MUCl, 2, 4, and 5AC (Table 2). In contrast, the clear cell tumor and mixed mesodermal tumors expressed only two to three mucins (Table 2). Mucin Gene Expression in Nonmalignant Ovarian Cell Lines. Normal ovaries do not have goblet cells or glands histologically. and, therefore, the normal tissue would not be expected to express secre tory mucin genes. Interestingly, the transformed, nonmalignant ovar ian epithelial cell lines expressed both MUC\ (an epithelial cell surface mucin) and MUC5AC (a secreted mucin). Other mucin genes were not expressed. Mucin Gene Expression with Advancing Tumor Stage. When mucin gene expression was compared to tumor stage, there was a significant decrease in expression of MUC3 and MUC4 as the tumor stage increased from I to IV, for all of the histological types (Fig. 2). When the subset of serous cystadenocarcinoma tumors were analyzed, a significant decrease in MUC4 expression with advancing stage was persistent (P < 0.05). Interestingly, the two RECs did not express MUC3 or MUC4. Mucin Gene Expression and Cancer Survival. The effect of mucin gene expression on survival was examined using Kaplan-Meier analysis. Although the differences in the survival curves were not statistically significant, a trend toward improved survival was noted in patients whose ovarian cancer tissues expressed MUC4 (Fig. 3).
DISCUSSION
To investigate the potential role of mucin glycoproteins in the metastatic process of ovarian cancers, we determined the profile of MUC genes expressed by ovarian tumors of different histological 5547
cell line from a
MUCIN GENE EXPRESSION
MUC4
1234
5
6
78
IN OVARIAN
EPITHELIAL
B
9 10 11 12 13 14 15 16 17 18
1
2
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3
4
5
6
7
8
9 10 11 12 13 14 15 16 17 18
MUC1
9.4-» 4.4-» 2.4-» 19 20 21 22 23 24 25 26 27
28 29 30 31 32 33 34 35
19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35
9.4-» 4.4-» 2.4-»
1 2
1 2
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34
56
7
8
9 10 11 12 13 14 15 16 17 18
t-••§!*»*tff*» 5
6
7
8
9 10 11 12 13 14 15 16 17 18
19 20 21 22 23 24 25 26 27 28 29 30 31 32 33
19 20 21 22 23 24 25 26 27 28 29 30 31 32 33
Fig. I. Northern analyses of MUC4 (A}. MUCl (B), and yactin (C) mRNA in ovarian cancer tissue samples. Total RNA ( 10 u,g), isolated from ovarian cancer tissue samples, CFPAC cells, and muscle tissue by single-step guanidine isothiocyanale-phenol-chloroforni method using TRlzol reagent, was blotted onto nylon membrane. RNA samples were arranged in order of advancing stage of the ovarian tumors (Lane numbers correspond to Table I ). Because of limited RNA quantities, samples 3, 13 and 16 were not evaluated for MUC4 expression and sample 26 was not evaluated for MUC\ expression. The two ovarian RECs are shown in Lanes 32-33. The positive and negative controls are a CFPAC (Lane 34) and normal human skeletal muscle (Lane 35}. Arrows in A and ßindicate RNA standards (kb); the arrow in C indicates the 2.4-kb RNA marker. D shows the elhidium stains of the Northern gels for all of the RNA samples.
types and stages. Although there was no significant correlation be tween specific mucin genes and histological types, a pattern emerged revealing that endometrioid adenocarcinoma, mucinous cystadenocarcinomas, and serous cystadenocarcinomas expressed at least four to five mucins, whereas the array of expression for mixed mesodermal tumors and clear cell adenocarcinoma was limited to two to three mucins. The pattern of mucin expression reported herein confirms previous reports of MUCl, 2, and 3 glycoprotein expression in normal and malignant ovarian cells. Ho el al. (25) determined by immunohisto-
Table 2 Mucin gene expression by histologie type MUC5BClear Histology Cell" Endometrioid Mucinous Serous MMT of the ovary
No. of samples 1 1 2 20 3
MUC\MUC2 + -I+ + -
" Clear cell, clear cell adenocarcinoma;
+ + + -
MUCi
MUC4
MUC5AC
+ +
+ + + +
+ + + +
+
-
+
Endometrioid, endometrioid adenocarcinoma;
Mucinous, mucinous cystadenocarcinoma; Serous, serous cysladenocarcinoma; MMT, mixed niesodermal tumor of the ovary; +. MUC gene expression is present in at least one sample; -, MUC gene expression is absent.
chemistry that although normal ovaries did not express any of the MUC epitopes, the mucinous cystadenocarcinomas expressed three mucin glycoproteins, MUCl, 2, and 3. This pattern of expression correlates closely with the expression of Mi/Ci, 2, and 3 mRNA by mucinous cystadenocarcinomas in our report. Tashiro el al. (12) reported that only MUCl glycoprotein was produced by the normal ovary while both MUCl and MUC2 glycoproteins were expressed by ovarian cancer. Dong et al. (24) reported that high levels of MUCl glycoprotein at the apical surface of ovarian cancers correlated with a poor prognosis whereas MUC2 glycoprotein expression had an in verse correlation with tumor grade. Hanski et al. (23) demonstrated that MUC2 mRNA was expressed in ovarian adenocarcinomas. Our study further expanded these findings to demonstrate that in addition to "intestinal" mucins (MUCl and 3; Ref. 35, 36), the ovarian cancers also expressed mucins found in the normal endocervix (MUC4, 5AC, and 5B: Ref. 33). These data suggest that ovarian epithelial cancers have the capacity to produce several different mucin glycoproteins during the metastatic process. Nonmalignant ovarian epithelial cells in culture expressed two mucin genes: MUC\ and 5AC. The presence of mRNA for a secretory mucin, A/Õ/C5AC,was surprising in these cells because normal ovar-
5548
MUCIN OHNE EXPRESSION
IN OVARIAN
ian epithelium contains a single epithelial cell layer without secretory cells. It will be important to determine whether normal ovarian cells produce mucin glycoproteins; if they do, mucins may function to prevent normal ovaries from adhering to other tissues. Interestingly, the nonmalignant cells did not express the other mucins that were expressed by ovarian cancer cells MUC2, 3, 4, and 5B. The change from negative to positive expression of these mucin genes in malig nant cells suggests that new onset expression of secretory mucin genes is associated with malignant transformation of the ovarian epithelium. A major finding in this report was the decrease in A/Õ/C3and MUC4 expression with advancing stages of cancer. There are several reports of decreasing MUC expression with invasive or metastatic tumors. In pancreatic cancer, MUC2 expression correlated with noninvasive but not with invasive tumors (18). Gastric cancers expressed MUC5AC glycoprotein with early invasion but not with late invasive cancer (20). Finally, in colon cancer, although MUC\ expression increased with métastases(37), MUC2 and MUC3 expression de creased with most forms of colon cancer (38). MUC2 and 5AC had decreased levels of expression in high-grade rectovillous adenoma compared with low-grade adenoma and were not present in colon adenocarcinoma (39). These studies indicate the importance of mucin expression at specific times during the metastatic process. Initially, mucin gene expression seems to increase as cancer cells disseminate to distant sites. Once cancer cells have successfully metastasized, however, they seem to lose the capacity to make mucin glycoprotein. To metastasize to distant sites, malignant cells must successfully complete a series of steps including: (a) dissociation from the primary tumor site; (b) localized invasion; (c) vascular and lymphatic spread; and (d) avid attachment to distant sites. In addition, malignant cells must escape immune surveillance. It is likely that mucins play an important role in all of these steps. Mucin glycoproteins function as cell-cell adhesion molecules, thereby mediating adhesion and migra tory capability of cells. Milkens el al. (3) demonstrated that alterations in mucins may decrease the ability of neoplastic cells to aggregate, thereby facilitating dissociation of tumor cells from the primary tumor site. Mucins that bind to basement membrane proteins (40) and to Land P-selectins (41) may play a role in adhesion and migration in colon cancer. Finally, alterations in mucin glycoproteins may enhance the ability of cancer cells to bind to the endothelium (42). Ovarian cancer cells can use similar mechanisms to disseminate throughout the peritoneum.
MUC 1234
MUC
MUC
MUC
MUC SAC
MUC SB
Fig. 2. Bar graph demonstraling mucin gene expression stratified by stage of disease for all of the ovarian cancer tissue samples except RECs. The number of samples for each stage follows: Stage I, n = 4 (D); Stage II, n = 6 (la); Stage HI. n = 16 (D); and Stage IV. n = 5 (•).Each bar represents the percenlage of samples posilive for mRNA expression of the MUC gene indicated (Mi/Ci, 2. 3. 4, 5AC. or 5B). ».significant trend for decreasing gene expression with increasing stage in MUC3 (P = 0.04) and MUC4 (P = 0.03). No statistically significant trends are noted for MUC\, 2. 5AC, or 5B.
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100 80 80
