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Overexpression of Cyclin D1 in Epithelial Ovarian Cancers
GYNECOLOGIC ONCOLOGY ARTICLE NO.
64, 189–195 (1997)
GO964569
Overexpression of Cyclin D1 in Epithelial Ovarian Cancers SALLY D. WORSLEY, M.SC., BRUCE A. J. PONDER, PH.D., FRCP,
AND
BARRY R. DAVIES, PH.D.1
Wellbeing Ovarian Cancer Research Centre, Department of Pathology, University of Cambridge, Box 238, Level 3 Laboratories Block, Addenbrookes Hospital, Hills Road, Cambridge CB2 2QQ, United Kingdom Received July 9, 1996
Amplification and overexpression of the cell cycle-related gene cyclin D1 have been demonstrated in several human malignancies and have been shown to be directly oncogenic in breast epithelium and lymphocytes. Overexpression of the gene can occur in the absence of gene amplification. We have investigated whether cyclin D1 is overexpressed in a panel of 43 sporadic epithelial ovarian cancers using immunohistochemistry. Cyclin D1 was overexpressed in 26% of these tumors. Overexpression of cyclin D1 is associated with borderline or well-differentiated, grade 1 tumors but does not correlate with a particular histological type, overexpression of the c-erb-B2 oncogene, or presence of estrogen receptors. It is suggested that overexpression of cyclin D1 may contribute to the pathogenesis of epithelial ovarian cancers, including a subset of tumors different from those overexpressing the c-erb-B2 oncogene. q 1997 Academic Press
INTRODUCTION
Cyclin D1, a member of a family of proteins that regulates the activity of cyclin-dependent protein kinases (cdks), has been demonstrated to act as an oncogene and has been implicated in the development of several human neoplasias [1]. Cyclins and cdks play a central role in control of the cell cycle; cyclin D1 is involved in controlling progression through G1 into S phase [2]. Inhibition of cyclin D1 function prevents cells from entering S phase [2–4], whereas overexpression of the gene has been shown to shorten the duration of G1 [5]. Normal mammalian cells have a checkpoint at the G1 –S boundary of the cell cycle to repair damaged DNA and hence prevent accumulation of mutations; defects in cell cycle control may, therefore, also enhance genetic instability [6, 7]. Indeed, cyclin D1 overexpression has been shown to enhance gene amplification [8]. In steroid-responsive tissues, regulation of cell proliferation occurs by cell cycle-specific actions on cells in G1 phase [9]. Hence steroids and steroid antagonists may regulate cell cycle progression by transcriptional regulation of cyclin expression. 1 To whom correspondence and reprint requests should be addressed. Fax: 01223 336902. E-mail: [email protected].
Cyclin D1 was isolated as the PRAD1 oncogene, which is clonally rearranged and transcriptionally activated in benign, monoclonal parathyroid adenomas [10], and is translocated and overexpressed in certain B-cell lymphomas [11]. The human cyclin D1 gene is located on chromosome 11q13; this locus has been shown to be amplified in several human cancers including breast and squamous cell [12], non-small cell lung, bladder, and esophageal cancers [13]. In breast cancer, 11q13 is amplified in 15 to 20% of tumors [14, 15] and overexpression of cyclin D1 has been demonstrated by immunohistochemistry in up to 80% of breast carcinomas [15–20]. Hence overexpression of cyclin D1 in breast cancer can occur even in the absence of gene amplification. Moreover, amplification and overexpression of cyclin D1 have been associated with a subset of estrogen receptor-positive breast tumors with a poor prognosis [20, 21]. Recently, overexpression of cyclin D1 mRNA has been shown to be a marker to distinguish invasive and in situ breast carcinomas from nonmalignant lesions [22], suggesting that deregulated expression of cyclin D1 is a common early event in mammary carcinogenesis. Studies in transgenic mice have confirmed that cyclin D1 can act as an oncogene and predispose animals to tumor development in the mammary glands [23] and lymphatic system [24]. Although amplification of the 11q13 locus has not been reported in ovarian cancer, expression of the adult folate receptor gene, which also maps to 11q13, has been shown to be upregulated in ovarian cancer cells in culture and in ovarian tumors [25]. Since overexpression of cyclin D1 in breast cancer can occur in the absence of 11q13 amplification, it is conceivable that cyclin D1 expression may also be upregulated in ovarian cancers. To investigate this, we have analyzed expression of cyclin D1 in a panel of 43 sporadic ovarian cancers by immunohistochemistry. Moreover, given that cyclin D1 may be regulated by steroids such as estrogen and can cause gene amplification we have also investigated whether (1) estrogen receptor status and (2) the expression of c-erb-B2, an oncogene that is frequently amplified and overexpressed in ovarian cancers, correlate with cyclin D1 expression in these tumors.
189
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TABLE 1 Expression of Cyclin D1, c-erb-B2, and Estrogen Receptors by Normal Ovarian Epithelium, Benign Cystadenomas, and Ovarian Cancers Cyclin D1a
c-erb-B2a
ERb
Tissue/tumor
//
/
0
//
/
0
/
{
0
Normal epithelium Cystadenomas Tumors
0/6 0/9 2/43
0/6 0/9 9/43
6/6 9/9 32/43
0/6 0/9 7/43
0/6 0/9 3/43
6/6 9/9 33/43
0/6 6/9d 11/43
6/6c 0/9 8/43
0/6 3/9d 24/43
a Expression was scored as follows: //, ú75% of cells stain; /, 10–70% of cells stain; 0, õ10% of cells stain. Only nuclear staining for cyclin D1 and membrane staining for c-erb-B2 was scored as positive. b Expression was scored as follows: /, ú50% of cells stain; {, 10–50% of cells stain; 0, õ10% of cells stain. Only nuclear staining for ERs was scored as positive. c Estrogen receptor positivity most frequent in clefts/inclusion cysts and areas of stratification. d All serous cystadenomas examined positive; all mucinous cystadenomas negative.
MATERIALS AND METHODS
Formalin-fixed, paraffin-embedded blocks of tumor samples from 43 patients presenting with primary ovarian cancer at St. Mary’s Hospital for Women and Children, Manchester, between 1978 and 1991 and University of Nottingham Medical School, Queens Medical Centre, Nottingham, between 1989 and 1991 were sent to our laboratory in Cambridge. In addition, nine tissue blocks of benign cystadenomas and six blocks of normal ovaries were selected from the archive at Addenbrookes Hospital, Cambridge. Tumor type and grade were determined by a histopathologist. A mouse monoclonal antibody raised against the human CCND1 gene product cyclin D1 [26] was obtained from Dr. Jiri Bartek, Danish Cancer Society. A mouse monoclonal antibody to the human estrogen receptor and a rabbit polyclonal antibody to a synthetic human c-erb-B2 peptide were obtained from Dako Ltd (High Wycombe, UK). Immunohistochemical staining was carried out using an indirect method employing an antibody–avidin/biotin complex horseradish peroxidase staining procedure [27]. All sections were incubated for 30 min in methanol, with hydrogen peroxide added to 0.05% (v/v) to block endogenous peroxidase activity. Antigen retrieval was carried out by highpressure heating in a pressure cooker [28] for staining with the cyclin D1 and estrogen receptor antibodies and by incubating in 0.05% (v/v) saponin for 30 min for staining with the c-erb-B2 antibody. Negative (first antibody excluded) and positive (breast carcinomas known to express the proteins at high levels) controls were included with each batch of slides to ensure consistency of staining intensity. Staining intensity was assessed independently by two persons (S.W. and B.D.) and any difference of opinion on initial evaluation was resolved by discussion. Statistical comparisons between tumor grade and cyclin D1 expression and between expression of the three gene products studied were made using the
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Fisher exact test. A P value less than 0.05 was considered significant. RESULTS
The level of expression of the cyclin D1, c-erb-B2, and estrogen receptor proteins in the 6 normal ovaries, 9 benign cystadenomas, and 43 ovarian carcinomas used in our study is presented in summary form in Table 1, and the level of expression of the proteins in each individual tumor is presented in Table 2. Overexpression of cyclin D1 was detected in 11 of 43 (26%) ovarian tumors stained (Tables 1 and 2). Expression was classified as positive if more than 10% of the tumor cells stained strongly in their nuclei. No expression was detected in normal ovarian surface epithelial cells (Fig. 1a), clefts, inclusion cysts, and benign cystadenomas (Fig. 1b). Strong expression of cyclin D1 in more than 75% of the epithelial tumor cells was only seen in two tumors (Tables 1 and 2); one of these was a well-differentiated mucinous tumor (Fig. 1c) and the other a well-differentiated mixed mucinous and endometrioid tumor. Variable expression was observed in a further 9 tumors (Table 2, Fig. 1d). Expression of cyclin D1 appeared to be associated with grade 1 tumors or lesions of borderline malignancy. When grade 1 and borderline tumors were compared with grade 2 and 3 tumors, a significantly greater number of borderline/grade 1 tumors were positive for cyclin D1 expression (Table 3). Overexpression of cyclin D1 was not confined to a particular histological type of tumor; at least one tumor of every histological type overexpressed this gene product (Table 4). The estrogen receptor status of the tumors was also determined. Estrogen receptor positivity was characterized by definite staining of the nucleus (Figs. 1e–g). Estrogen receptors were present in more than 50% of cells in a total of 11 of 43 (26%) tumors. A further 8 of 43 (19%) tumors posessed between 10 and 50% of estrogen receptor-positive cells,
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CYCLIN D1 OVEREXPRESSION IN OVARIAN CANCER
TABLE 2 Expression of Cyclin D1, c-erb-B2, and Estrogen Receptors in Individual Ovarian Carcinomas Tumor histology
Grade
Cyclin D1a
c-erb-B2b
ERsc
Mucinous Mucinous Endometrioid Mucinous Mucinous Mucinous Mucinous Mucinous Clear cell Clear cell Endometrioid Endometrioid Endometrioid Endometrioid Endometrioid Endometrioid Endometrioid Mixed Mixed Mixed Mixed Serous Serous Clear cell Clear cell Clear cell Endometrioid Endometrioid Endometrioid Endometrioid Endometrioid Mixed Serous Serous Serous Serous Serous Poorly differentiated Poorly differentiated Poorly differentiated Poorly differentiated Serous Endometrioid
Borderline Borderline Borderline 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 2 2 2 2 2 2 2 2 2 2 2 3 3 3 3 3 3 3 3 3 Varies 1–3 1–2
/ 0 / 0 0 // / 0 / 0 0 0 / 0 0 0 / 0 // 0 0 0 0 0 / 0 0 0 0 0 0 0 0 0 0 0 0 0 0 / 0 / 0
0 0 0 // 0 /// 0 / 0 // 0 0 0 0 0 0 0 0 0 0 0 / 0 0 0 0 0 0 0 0 // 0 // 0 0 0 0 0 / 0 // 0 //
0 0 0 0 { 0 0 0 0 0 / { 0 { 0 / / 0 0 / / 0 / 0 0 0 / 0 0 / { 0 { { { / / { 0 / 0 0 0
a
Expression was scored as follows: //, ú75% of cells stain; /, 10– 70% of cells stain; 0, õ10% of cells stain. Only nuclear staining for cyclin D1 is scored as positive. b Expression was scored as follows: ///, ú75% of cells stain strongly, //, ú75% of cells stain variably or weakly; /, 10–70% of cells stain to some extent; 0, no cells stain. Only membrane staining for c-erb-B2 was scored as positive. c Expression was scored as follows: /, ú50% of cell stain; {, 10–50% of cells stain; 0, õ10% of cells stain. Only nuclear staining for ERs was scored as positive.
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while 24 of 43 (56%) tumors possessed less than 10% of estrogen receptor-positive cells (Tables 1 and 2). Only tumors that possessed more than 50% of estrogen receptorpositive cells were classified as estrogen receptor positive. Interestingly, all benign mucinous cystadenomas and mucinous carcinomas were estrogen receptor negative. Normal ovarian surface epithelium showed variable staining, but estrogen receptor positivity was more common in clefts, inclusion cysts, and where stratification of the epithelium occurred. Definite consistent membrane staining for c-erb-B2 was seen in 7 of 43 (16%) tumors in our study. The staining intensity varied from weak to very intense (Figs. 1h–j). Variable expression of c-erb-B2 was seen in a further 3 tumors. Therefore, 10 of 43 (23%) tumors were classified as positive for c-erb-B2 expression (Tables 1 and 2). Expression of c-erb-B2 was not detectable in inclusion cysts, clefts, and normal surface epithelial cells of the ovary. In contrast to cyclin D1 expression, no correlation was apparent between c-erb-B2 expression and tumor grade, although none of the three tumors of borderline malignancy overexpressed this gene product (Table 3). Expression of c-erb-B2 was not limited to poorly differentiated tumors. No correlation was observed between histological type and c-erb-B2 expression (Table 4). Although the numbers were small, we attempted to determine if there was a correlation between cyclin D1 expression and estrogen receptor status or c-erb-B2 expression (Table 5). Because of the small sample size, however, correlation or lack of correlation between expression of the gene products analyzed should not be regarded as definitive. Of the 11 tumors that showed overexpression of the cyclin D1 protein, only one of these tumors also stained with antiserum to cerb-B2. This was a well-differentiated mucinous tumor. A total of 20 of 43 (47%) tumors showed expression of either one or both of the two proteins, but only 1 of 43 expressed both; however, this apparent correlation between c-erb-B2 overexpression and lack of cyclin D1 expression did not achieve statistical significance (P Å 0.162). No correlation was found to exist between estrogen receptor status and cyclin D1 overexpression (P Å 0.269); however, a significant correlation was found between overexpression of c-erb-B2 and absence of estrogen receptors when tumors containing less than 50% positive cells were classified as negative for estrogen receptor (P Å 0.034). None of the 10 tumors showing overexpression of c-erb-B2 expressed estrogen receptors in more than 50% of the tumor cells (Table 5). DISCUSSION
In this study, we report for the first time that the cell cycle-associated protein cyclin D1 is overexpressed in approximately one-quarter (26%) of sporadic epithelial ovarian
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FIG. 1. Immunocytochemical staining of ovarian tumors with antibodies to cyclin D1, c-erb-B2, and estrogen receptor. (a) Surface epithelial cells (arrows) of the normal ovary fail to stain with antiserum to cyclin D1. (b) Epithelial cells in a mucinous cystadenoma fail to stain with antiserum to cyclin D1. (c) Epithelial cells in a well-differentiated mucinous carcinoma show strong nuclear staining with antiserum to cyclin D1. The stromal component of the tumor fails to stain. (d) A well-differentiated endometrioid tumor showing variable nuclear staining with cyclin D1 antiserum. (e) Endometrioid carcinoma showing strong nuclear staining with antiserum to estrogen receptor. (f) Serous papillary adenocarcinoma showing moderate uniform expression of estrogen receptor. (g) A serous papillary carcinoma different from that shown in (f) fails to stain with antiserum to estrogen receptor. (h) A poorly differentiated carcinoma showing weak membrane staining with antiserum to c-erb-B2. (i) Endometrioid carcinoma showing strong membrane staining with c-erb-B2 antiserum. Note that the stromal component of the tumor (center) fails to stain. (j) The same well-differentiated mucinous tumor as shown in (c) also stains strongly with antiserum to c-erb-B2. Again, only the epithelial cells stain. Magnification of all photographs, 140.
tumors. This number is roughly comparable to the number of tumors overexpressing the c-erb-B2 oncogene both in our study (23%) and in the 20 to 30% reported in other studies [29–31]; however, the spectra of tumors overexpressing these two oncoproteins are very different. With one excep-
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tion, tumors expressing cyclin D1 do not express c-erb-B2. This exception was a well-differentiated mucinous tumor. This suggests that, in general, cyclin D1 is overexpressed in a subset of ovarian tumors different from those that overexpress the c-erb-B2 oncogene.
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CYCLIN D1 OVEREXPRESSION IN OVARIAN CANCER
FIG. 1—Continued
In our study a correlation was found between tumor grade and cyclin D1 overexpression. Cyclin D1 expression is more commonly found in borderline tumors and grade 1 carcinomas than in grade 2 and 3 carcinomas. There are a number of possible interpretations of this result. A higher frequency of cyclin D1 overexpression in borderline and lower-grade tumors suggests that deregulation of cyclin D1 expression may be a relatively early event in the pathogenesis of epitheTABLE 3 Relationship between Tumor Grade and Expression of Cyclin D1, c-erb-B2, and Estrogen Receptors Tumor grade
Cyclin D1 positivea
ER positiveb
c-erb-B2 positivec
2/3
0/3
0/3
5/18 3/10
4/18 2/10
3/10 0/2
3/10 1/2
Borderline
8/21d Grade 1 Grade 2
6/18 1/10
Grade 3 Mixed
1/10 1/2
lial ovarian cancer. In contrast, overexpression of the c-erbB2 gene may generally be a later event; a correlation with poor prognosis has been associated with overexpression of this oncogene in some studies [29–31]; however, it is possible that many of the borderline/low-grade, cyclin D1-overexpressing tumors are distinctly different lesions that have a pathogenesis different from that of many higher-grade tumors. Other types of mutation that result in higher-grade tumors may be underrepresented in borderline/lower-grade tumors. Further studies in which parameters such as stage and survival are included are clearly necessary to gain a better understanding of the role that cyclin D1 plays in ovarian cancer and its possible use as a prognostic indicator. TABLE 4 Relationship between Tumor Histology and Expression of Cyclin D1, c-erb-B2, and Estrogen Receptors
2/20d
a
Tumors with ú10% positive nuclei. Tumors with ú50% positive nuclei. c Tumors with some or all cells showing definite membrane staining. d Significantly more tumors borderline/Grade 1 cyclin D1 positive than grade 2/grade 3 cyclin D1 positive (P Å 0.035, Fisher exact test). b
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Tumor histology
Cyclin D1 positive
c-erb-B2 positive
ER positive
Serous Mucinous Endometrioid Clear cell Poorly differentiated
1/8 3/7 3/14 2/5 1/4
2/8 3/7 2/14 1/5 2/4
3/8 0/7 5/14 0/5 1/4
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TABLE 5 Analysis of Combinations of Gene Products in Ovarian Tumors Combination Cyclin Cyclin Cyclin Cyclin
Number of tumors
D1//c-erb-B2/ D1//c-erb-B20 D10/c-erb-B2/ D10/c-erb-B20
1/43 10/43 9/43 23/43 P Å 0.162
Cyclin Cyclin Cyclin Cyclin
D1//ER/ D1//ER0 D10/ER/ D10/ER0
2/43 9/43 9/43 23/43 P Å 0.269
c-erb-B2//ER/ c-erb-B2//ER0 c-erb-B20/ER/ c-erb-B20/ER0
0/43 10/43 11/43 22/43 P Å 0.034
Furthermore, the development of appropriate animal models for epithelial ovarian cancer will allow more revealing functional studies to be carried out to determine the phenotypic consequences of cyclin D1 overexpression in ovarian epithelium. Cyclin D1 has been shown to enhance amplification of the CAD gene by more than 1000-fold in rat liver epithelial cells, apparently due to loss of control of the G1 –S cell cycle checkpoint [8]. Although c-erb-B2 is frequently amplified and overexpressed in ovarian cancers, its expression does not appear to be deregulated by cyclin D1 expression. In our study, c-erb-B2 expression did not correlate with tumor grade but did correlate with absence of estrogen receptors. The frequency of ovarian tumors showing overexpression of cyclin D1 in our study is rather lower than the reported frequencies of breast and certain other cancers [17, 32] showing overexpression of this protein. It is only possible to speculate why this may be the case. First, there are almost certainly multiple pathways to tumor development and cyclin D1 overexpression may contribute to only one or a proportion of these pathways. Second, it is probable that the cyclin D1 protein is functionally redundant and that other proteins can substitute for its contribution to a carcinogenic pathway. Cancer cells may use other mechanisms for cell cycle progression from G1 to S phase. For example, mutation or downregulation of expression of the multiple tumor suppressor gene (MTS1) may substitute for cyclin D1 overexpression. Moreover, an absence of a requirement for cyclin D1 has been shown in breast cancer cell lines with defects in Rb function [33]. This having been said, genetic changes to MTS1 appear to be infrequent in ovarian cancer [34, 35]; similarly genetic changes to the Rb gene and abnormal Rb gene expression have also been reported to be uncommon
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in ovarian cancer [36–38]. Whatever the explanation, it is clear that cyclin D1 overexpression is not an obligatory requirement for the proliferation of epithelial ovarian cancers. There has been some suggestion that deregulation of cyclin D1 expression is also an early event in the pathogenesis of breast cancer. When a comparison was made between matched lesions at different stages of tumor progression, cyclin D1 mRNA expression was found to be an early event that is maintained throughout progression including metastatic spread [22]. This suggests that cyclin D1-expressing breast cancers need to retain expression of this gene product to develop into a fully invasive, metastatic cancer. It would be interesting to determine whether this is the case in epithelial ovarian cancer. In breast cancer cells, cyclin D1 expression has been shown to be induced by steroids and its expression inhibited by antiestrogens. Therefore, we investigated whether overexpression of cyclin D1 in ovarian tumors required the presence of estrogen receptors. In our study, we found no correlation between cyclin D1 expression and the presence or absence of estrogen receptors. Clearly a level of estrogen receptor expression detectable by immunocytochemistry is not a requirement for deregulated cyclin D1 expression in ovarian cancers. In conclusion, cyclin D1 overexpression is found in approximately one-quarter of ovarian cancers. Overexpression of this gene occurs predominantly in tumors that do not overexpress the c-erb-B2 oncogene, but does not correlate with the presence of estrogen receptors. The functional consequences of deregulated cyclin D1 expression in ovarian epithelium and its relationship to patient prognosis deserve further investigation. ACKNOWLEDGMENTS The authors thank Dr. Hilary Buckley (St. Mary’s Hospital, Manchester, UK) and Dr. M. C. Anderson (Queens Medical Centre, University of Nottingham Medical School) for providing some of the tumor samples and pathology reports. This work was funded by WellBeing.
REFERENCES 1. Draetta G: Mammalian G1 cyclins. Curr Opin Cell Biol 6:8429–8460, 1994 2. Baldin V, Lukas J, Marcote MJ, Pagano M, Draetta G: Cyclin D1 is a nuclear protein required for cell cycle progression in G1 . Genes Dev 7:812–821, 1993 3. Quelle D, Ashmun R, Shurtleff S, Kato J, Bar-Sagi D, Roussel M, Sherr C: Overexpression of mouse D-type cyclins accelerates G1 phase in rodent fibroblasts. Genes Dev 7:1559–1571, 1993 4. Lukas J, Pagano M, Staskova Z, Draetta G, Bartek J: Cyclin D1 protein oscillates and is essential for cell cycle progression in human tumor cell lines. Oncogene 9:707–718, 1994 5. Jiang W, Khan SM, Zhou P, Zhang YJ, Cacace AM, Infante AS: Overexpression of cyclin D1 in rat fibroblasts causes abnormalities in
goas
AP: GYN
CYCLIN D1 OVEREXPRESSION IN OVARIAN CANCER growth control, cell cycle progression and gene expression. Oncogene 8:3447–3457, 1993 6. Hartwell L: Defects in cell cycle checkpoints may be responsible for the genomic instability of cancer cells. Cell 71:543–546, 1992 7. Almason A, Linke SP, Paulson TG, Wall GM: Genetic instability as a consequence of inappropriate entry into the cell cycle progression through S phase. Cancer Metast Rev 14:59–73, 1995 8. Zhou P, Jiang W, Weghorst CM, Weinstein IB: Overexpression of cyclin D1 enhances gene amplification. Cancer Res 56:36–39, 1996 9. Sutherland RL, Hamilton JA, Sweeney KJE, Watts CKW, Musgrove EA: Expression and regulation of cyclin genes in breast cancer. Acta Oncol 34:651–656, 1995 10. Arnold A, Kim HG, Gaz RD, Eddy RL, Fukusima Y, Kronenberg HM: Molecular cloning and chromosomal mapping of DNA rearranged with the parathyroid hormone gene in a parathyroid adenoma. J Clin Invest 83:2034–2040, 1989 11. Williams ME, Swerdlow SH, Rosenberg CL, Arnold A: Centrocytic lymphoma: A B-cell non-Hodgkin’s lymphoma characterised by chromosome 11 bcl-1 and PRAD1 rearrangements. Curr Top Microbiol Immunol 182:325–329, 1992
23.
24.
25.
26.
27.
28.
12. Brookes S, Lammie GA, Schuuring E, Dickson C, Peters G: Linkage map of a region of human chromosome band 11q13 amplified in breast and squamous cell tumors. Genes Chromosom Cancer 4:290–301, 1992
29.
13. Jiang W, Kahn SM, Tomita N, Zhang YJ, Lu SH, Weinstein IB: Amplification and expression of the human cyclin D1 gene in oesophageal cancer. Cancer Res 52:2980–2983, 1992
30.
14. Lammie GA, Peters G: Chromosome 11q13 abnormalities in human cancer. Cancer Cells 3:413–419, 1991 15. Champeme MH, Bieche I, Lizard S, Liderau R: 11q13 amplification in local recurrence of human primary breast cancer. Genes Chromosom Cancer 12:128–133, 1995
31.
16. Buckley MF, Sweeney KJE, Hamilton JA, Sini RL, Manning DL, Nicholson RI, deFaxio A, Watts CKW, Musgrove EA, Sutherland RL: Expression and amplification of cyclin genes in human breast cancer. Oncogene 8:2127–2133, 1993
32.
17. Gillet C, Frantl V, Smith R, Fisher C, Bartek J, Dickson C, Barnes D, Peters G: Amplification and overexpression of cyclin D1 in breast cancer detected by immunocytochemical staining. Cancer Res 54:1812–1817, 1994 18. Bartkova J, Lukas J, Muller H, Lutzhoft D, Strauss M, Bartek J: Cyclin D1 protein expression and function in human breast cancer. Int J Cancer 57:353–361, 1994
33.
34.
35.
19. Worsley SD, Jennings BA, Khalil KH, Mole M, Girling AC: Cyclin D1 amplification and expression in human breast carcinoma: Correlation with histological prognostic markers and oestrogen receptor expression. J Clin Pathol 49:M46–M50, 1996
36.
20. Michalides R, Hageman P, van Tinteren H, Houben L, Weintjens E, Klompmaker R, Pterse J: A clinicopathological study on overexpression of cyclin D1 and of p53 in a series of 248 patients with operable breast cancer. Br J Cancer 73:728–734, 1996
37.
21. Fantl V, Richards MA, Smith R: Gene amplification on chromosome band 11q13 and estrogen receptor status in breast cancer. Eur J Cancer 26:423–429, 1990 22. Weinstat-Saslow D, Merlino MJ, Manrow RE, Lawrence JA, Bluth RF, Wittenbel KD, Simpson JF, Page DL, Steeg PS: Overexpression of
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38.
195
cyclin D mRNA distinguishes in situ and invasive breast carcinomas from non-malignant lesions. Nature Med 1:1256–1260, 1996 Wang TC, Cardiff RD, Zukerberg L, Lees E, Arnold A, Schmidt EV: Mammary hyperplasia and carcinoma in MMTV–cyclin D1 transgenic mice. Nature 369:669–671, 1994 Bodrug SE, Warner BJ, Bath ML, Lindeman GJ, Harris AW, Adams JM: Cyclin D1 transgene impedes lymphocyte maturation and collaborates in lymphoma genesis with the myc gene. EMBO J 13:2124–2130, 1994 Campbell IG, Jones TA, Foulkes WD, Trowsdale J: Folate binding protein is a marker for ovarian cancer. Cancer Res 51:5329–5338, 1991 Hsu SM, Raine L, Fanger H: Use of avidin–biotin–peroxidase complex (ABC) in immunoperoxidase techniques. J Histochem Cytochem 29:577, 1981 Norton AJ, Jordan S, Yeomans P: Brief, high temperature heat denaturation (pressure cooking): A simple and effective method of antigen retrieval for routinely processed tissues. J Clin Pathol 173:371–379, 1994 Berchuck A, Kamel A, Whitaker R, Kerns B, Olt G, Kinney R, Soper T, Dodge R, Clarke-Pearson P, McKenzie S, Yin S, Bast RC: Overexpression of HER-2/neu is associated with poor survival in advanced epithelial ovarian cancer. Cancer Res 50:4087–4091, 1990 Slamon DJ, Godolphin W, Jones LA, et al.: Studies of HER-2/neu proto-oncogene in human breast and ovarian cancer. Science 244:707– 712, 1989 Felip E, Del Campo JM, Rubio D, Vidal MT, Colomer R, Bermejo B: Overexpression of c-erb-B2 in epithelial ovarian cancer. Cancer 75:2147–2152, 1995 Zhang S-Y, Caamano J, Cooper F, Guo X, Klein-Szanto AJP: Immunohistochemistry of cyclin D1 in human breast cancer. Am J Clin Pathol 102:695–698, 1994 Zhang S-Y, Caamano J, Cooper F, Guo X, Klein-Szanto AJP: Immunohistochemistry of cyclin D1 in human breast cancer. Am J Clin Pathol 102:695–698, 1994 Lukas J, Bartkova J, Rohde M, Strauss M, Bartek J: Cyclin D1 is dispensible for G1 control in retinoblastoma gene-deficient cells independent of cdk4 activity. Mol Cell Biol 15:2600–2611, 1995 Rodabaugh KJ, Biggs RB, Qureshi JA, Barrett AJ, Welch WR, Bell DA, Berkowitz RS, Mok SC: Detailed deletion mapping of chromosome 9p and p16 gene alterations in human borderline and invasive epithelial ovarian tumors. Oncogene 11:1249–1254, 1995 Campbell IG, Beynon G, Davis M, Englefield P: LOH and mutation analysis of CDKN2 in primary human ovarian cancers. Int J Cancer 63:222–225, 1995 Dodson MK, Cliby WA, Xu HJ, DeLacey KA, Hu SX, Keeney GL, Li J, Podratz KC, Jenkins RB, Benedict WF: Evidence of functional Rb protein in epithelial ovarian carcinomas despite loss of heterozygosity at the Rb locus. Cancer Res 54:610–613, 1994 Kim TM, Benedict WF, Xu HJ, Hu SX, Gosewehr J, Velicescu M, Yin E, Zheng J, D’Ablaing G, Dubeau L: Loss of heterozygosity on chromosome 13 is common only in the biologically more aggressive subtypes of ovarian epithelial tumors and is associated with normal retinoblastoma gene expression. Cancer Res 54:605–609, 1994 Liu Y, Heyman M, Wang Y, Falkmer IJ, Hising C, Szekely L, Einhorn S: Molecular analysis of the retinoblastoma gene in primary ovarian cancer cells. Int J Cancer 58:663–667, 1994
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GO964569
Overexpression of Cyclin D1 in Epithelial Ovarian Cancers SALLY D. WORSLEY, M.SC., BRUCE A. J. PONDER, PH.D., FRCP,
AND
BARRY R. DAVIES, PH.D.1
Wellbeing Ovarian Cancer Research Centre, Department of Pathology, University of Cambridge, Box 238, Level 3 Laboratories Block, Addenbrookes Hospital, Hills Road, Cambridge CB2 2QQ, United Kingdom Received July 9, 1996
Amplification and overexpression of the cell cycle-related gene cyclin D1 have been demonstrated in several human malignancies and have been shown to be directly oncogenic in breast epithelium and lymphocytes. Overexpression of the gene can occur in the absence of gene amplification. We have investigated whether cyclin D1 is overexpressed in a panel of 43 sporadic epithelial ovarian cancers using immunohistochemistry. Cyclin D1 was overexpressed in 26% of these tumors. Overexpression of cyclin D1 is associated with borderline or well-differentiated, grade 1 tumors but does not correlate with a particular histological type, overexpression of the c-erb-B2 oncogene, or presence of estrogen receptors. It is suggested that overexpression of cyclin D1 may contribute to the pathogenesis of epithelial ovarian cancers, including a subset of tumors different from those overexpressing the c-erb-B2 oncogene. q 1997 Academic Press
INTRODUCTION
Cyclin D1, a member of a family of proteins that regulates the activity of cyclin-dependent protein kinases (cdks), has been demonstrated to act as an oncogene and has been implicated in the development of several human neoplasias [1]. Cyclins and cdks play a central role in control of the cell cycle; cyclin D1 is involved in controlling progression through G1 into S phase [2]. Inhibition of cyclin D1 function prevents cells from entering S phase [2–4], whereas overexpression of the gene has been shown to shorten the duration of G1 [5]. Normal mammalian cells have a checkpoint at the G1 –S boundary of the cell cycle to repair damaged DNA and hence prevent accumulation of mutations; defects in cell cycle control may, therefore, also enhance genetic instability [6, 7]. Indeed, cyclin D1 overexpression has been shown to enhance gene amplification [8]. In steroid-responsive tissues, regulation of cell proliferation occurs by cell cycle-specific actions on cells in G1 phase [9]. Hence steroids and steroid antagonists may regulate cell cycle progression by transcriptional regulation of cyclin expression. 1 To whom correspondence and reprint requests should be addressed. Fax: 01223 336902. E-mail: [email protected].
Cyclin D1 was isolated as the PRAD1 oncogene, which is clonally rearranged and transcriptionally activated in benign, monoclonal parathyroid adenomas [10], and is translocated and overexpressed in certain B-cell lymphomas [11]. The human cyclin D1 gene is located on chromosome 11q13; this locus has been shown to be amplified in several human cancers including breast and squamous cell [12], non-small cell lung, bladder, and esophageal cancers [13]. In breast cancer, 11q13 is amplified in 15 to 20% of tumors [14, 15] and overexpression of cyclin D1 has been demonstrated by immunohistochemistry in up to 80% of breast carcinomas [15–20]. Hence overexpression of cyclin D1 in breast cancer can occur even in the absence of gene amplification. Moreover, amplification and overexpression of cyclin D1 have been associated with a subset of estrogen receptor-positive breast tumors with a poor prognosis [20, 21]. Recently, overexpression of cyclin D1 mRNA has been shown to be a marker to distinguish invasive and in situ breast carcinomas from nonmalignant lesions [22], suggesting that deregulated expression of cyclin D1 is a common early event in mammary carcinogenesis. Studies in transgenic mice have confirmed that cyclin D1 can act as an oncogene and predispose animals to tumor development in the mammary glands [23] and lymphatic system [24]. Although amplification of the 11q13 locus has not been reported in ovarian cancer, expression of the adult folate receptor gene, which also maps to 11q13, has been shown to be upregulated in ovarian cancer cells in culture and in ovarian tumors [25]. Since overexpression of cyclin D1 in breast cancer can occur in the absence of 11q13 amplification, it is conceivable that cyclin D1 expression may also be upregulated in ovarian cancers. To investigate this, we have analyzed expression of cyclin D1 in a panel of 43 sporadic ovarian cancers by immunohistochemistry. Moreover, given that cyclin D1 may be regulated by steroids such as estrogen and can cause gene amplification we have also investigated whether (1) estrogen receptor status and (2) the expression of c-erb-B2, an oncogene that is frequently amplified and overexpressed in ovarian cancers, correlate with cyclin D1 expression in these tumors.
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TABLE 1 Expression of Cyclin D1, c-erb-B2, and Estrogen Receptors by Normal Ovarian Epithelium, Benign Cystadenomas, and Ovarian Cancers Cyclin D1a
c-erb-B2a
ERb
Tissue/tumor
//
/
0
//
/
0
/
{
0
Normal epithelium Cystadenomas Tumors
0/6 0/9 2/43
0/6 0/9 9/43
6/6 9/9 32/43
0/6 0/9 7/43
0/6 0/9 3/43
6/6 9/9 33/43
0/6 6/9d 11/43
6/6c 0/9 8/43
0/6 3/9d 24/43
a Expression was scored as follows: //, ú75% of cells stain; /, 10–70% of cells stain; 0, õ10% of cells stain. Only nuclear staining for cyclin D1 and membrane staining for c-erb-B2 was scored as positive. b Expression was scored as follows: /, ú50% of cells stain; {, 10–50% of cells stain; 0, õ10% of cells stain. Only nuclear staining for ERs was scored as positive. c Estrogen receptor positivity most frequent in clefts/inclusion cysts and areas of stratification. d All serous cystadenomas examined positive; all mucinous cystadenomas negative.
MATERIALS AND METHODS
Formalin-fixed, paraffin-embedded blocks of tumor samples from 43 patients presenting with primary ovarian cancer at St. Mary’s Hospital for Women and Children, Manchester, between 1978 and 1991 and University of Nottingham Medical School, Queens Medical Centre, Nottingham, between 1989 and 1991 were sent to our laboratory in Cambridge. In addition, nine tissue blocks of benign cystadenomas and six blocks of normal ovaries were selected from the archive at Addenbrookes Hospital, Cambridge. Tumor type and grade were determined by a histopathologist. A mouse monoclonal antibody raised against the human CCND1 gene product cyclin D1 [26] was obtained from Dr. Jiri Bartek, Danish Cancer Society. A mouse monoclonal antibody to the human estrogen receptor and a rabbit polyclonal antibody to a synthetic human c-erb-B2 peptide were obtained from Dako Ltd (High Wycombe, UK). Immunohistochemical staining was carried out using an indirect method employing an antibody–avidin/biotin complex horseradish peroxidase staining procedure [27]. All sections were incubated for 30 min in methanol, with hydrogen peroxide added to 0.05% (v/v) to block endogenous peroxidase activity. Antigen retrieval was carried out by highpressure heating in a pressure cooker [28] for staining with the cyclin D1 and estrogen receptor antibodies and by incubating in 0.05% (v/v) saponin for 30 min for staining with the c-erb-B2 antibody. Negative (first antibody excluded) and positive (breast carcinomas known to express the proteins at high levels) controls were included with each batch of slides to ensure consistency of staining intensity. Staining intensity was assessed independently by two persons (S.W. and B.D.) and any difference of opinion on initial evaluation was resolved by discussion. Statistical comparisons between tumor grade and cyclin D1 expression and between expression of the three gene products studied were made using the
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Fisher exact test. A P value less than 0.05 was considered significant. RESULTS
The level of expression of the cyclin D1, c-erb-B2, and estrogen receptor proteins in the 6 normal ovaries, 9 benign cystadenomas, and 43 ovarian carcinomas used in our study is presented in summary form in Table 1, and the level of expression of the proteins in each individual tumor is presented in Table 2. Overexpression of cyclin D1 was detected in 11 of 43 (26%) ovarian tumors stained (Tables 1 and 2). Expression was classified as positive if more than 10% of the tumor cells stained strongly in their nuclei. No expression was detected in normal ovarian surface epithelial cells (Fig. 1a), clefts, inclusion cysts, and benign cystadenomas (Fig. 1b). Strong expression of cyclin D1 in more than 75% of the epithelial tumor cells was only seen in two tumors (Tables 1 and 2); one of these was a well-differentiated mucinous tumor (Fig. 1c) and the other a well-differentiated mixed mucinous and endometrioid tumor. Variable expression was observed in a further 9 tumors (Table 2, Fig. 1d). Expression of cyclin D1 appeared to be associated with grade 1 tumors or lesions of borderline malignancy. When grade 1 and borderline tumors were compared with grade 2 and 3 tumors, a significantly greater number of borderline/grade 1 tumors were positive for cyclin D1 expression (Table 3). Overexpression of cyclin D1 was not confined to a particular histological type of tumor; at least one tumor of every histological type overexpressed this gene product (Table 4). The estrogen receptor status of the tumors was also determined. Estrogen receptor positivity was characterized by definite staining of the nucleus (Figs. 1e–g). Estrogen receptors were present in more than 50% of cells in a total of 11 of 43 (26%) tumors. A further 8 of 43 (19%) tumors posessed between 10 and 50% of estrogen receptor-positive cells,
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CYCLIN D1 OVEREXPRESSION IN OVARIAN CANCER
TABLE 2 Expression of Cyclin D1, c-erb-B2, and Estrogen Receptors in Individual Ovarian Carcinomas Tumor histology
Grade
Cyclin D1a
c-erb-B2b
ERsc
Mucinous Mucinous Endometrioid Mucinous Mucinous Mucinous Mucinous Mucinous Clear cell Clear cell Endometrioid Endometrioid Endometrioid Endometrioid Endometrioid Endometrioid Endometrioid Mixed Mixed Mixed Mixed Serous Serous Clear cell Clear cell Clear cell Endometrioid Endometrioid Endometrioid Endometrioid Endometrioid Mixed Serous Serous Serous Serous Serous Poorly differentiated Poorly differentiated Poorly differentiated Poorly differentiated Serous Endometrioid
Borderline Borderline Borderline 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 2 2 2 2 2 2 2 2 2 2 2 3 3 3 3 3 3 3 3 3 Varies 1–3 1–2
/ 0 / 0 0 // / 0 / 0 0 0 / 0 0 0 / 0 // 0 0 0 0 0 / 0 0 0 0 0 0 0 0 0 0 0 0 0 0 / 0 / 0
0 0 0 // 0 /// 0 / 0 // 0 0 0 0 0 0 0 0 0 0 0 / 0 0 0 0 0 0 0 0 // 0 // 0 0 0 0 0 / 0 // 0 //
0 0 0 0 { 0 0 0 0 0 / { 0 { 0 / / 0 0 / / 0 / 0 0 0 / 0 0 / { 0 { { { / / { 0 / 0 0 0
a
Expression was scored as follows: //, ú75% of cells stain; /, 10– 70% of cells stain; 0, õ10% of cells stain. Only nuclear staining for cyclin D1 is scored as positive. b Expression was scored as follows: ///, ú75% of cells stain strongly, //, ú75% of cells stain variably or weakly; /, 10–70% of cells stain to some extent; 0, no cells stain. Only membrane staining for c-erb-B2 was scored as positive. c Expression was scored as follows: /, ú50% of cell stain; {, 10–50% of cells stain; 0, õ10% of cells stain. Only nuclear staining for ERs was scored as positive.
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while 24 of 43 (56%) tumors possessed less than 10% of estrogen receptor-positive cells (Tables 1 and 2). Only tumors that possessed more than 50% of estrogen receptorpositive cells were classified as estrogen receptor positive. Interestingly, all benign mucinous cystadenomas and mucinous carcinomas were estrogen receptor negative. Normal ovarian surface epithelium showed variable staining, but estrogen receptor positivity was more common in clefts, inclusion cysts, and where stratification of the epithelium occurred. Definite consistent membrane staining for c-erb-B2 was seen in 7 of 43 (16%) tumors in our study. The staining intensity varied from weak to very intense (Figs. 1h–j). Variable expression of c-erb-B2 was seen in a further 3 tumors. Therefore, 10 of 43 (23%) tumors were classified as positive for c-erb-B2 expression (Tables 1 and 2). Expression of c-erb-B2 was not detectable in inclusion cysts, clefts, and normal surface epithelial cells of the ovary. In contrast to cyclin D1 expression, no correlation was apparent between c-erb-B2 expression and tumor grade, although none of the three tumors of borderline malignancy overexpressed this gene product (Table 3). Expression of c-erb-B2 was not limited to poorly differentiated tumors. No correlation was observed between histological type and c-erb-B2 expression (Table 4). Although the numbers were small, we attempted to determine if there was a correlation between cyclin D1 expression and estrogen receptor status or c-erb-B2 expression (Table 5). Because of the small sample size, however, correlation or lack of correlation between expression of the gene products analyzed should not be regarded as definitive. Of the 11 tumors that showed overexpression of the cyclin D1 protein, only one of these tumors also stained with antiserum to cerb-B2. This was a well-differentiated mucinous tumor. A total of 20 of 43 (47%) tumors showed expression of either one or both of the two proteins, but only 1 of 43 expressed both; however, this apparent correlation between c-erb-B2 overexpression and lack of cyclin D1 expression did not achieve statistical significance (P Å 0.162). No correlation was found to exist between estrogen receptor status and cyclin D1 overexpression (P Å 0.269); however, a significant correlation was found between overexpression of c-erb-B2 and absence of estrogen receptors when tumors containing less than 50% positive cells were classified as negative for estrogen receptor (P Å 0.034). None of the 10 tumors showing overexpression of c-erb-B2 expressed estrogen receptors in more than 50% of the tumor cells (Table 5). DISCUSSION
In this study, we report for the first time that the cell cycle-associated protein cyclin D1 is overexpressed in approximately one-quarter (26%) of sporadic epithelial ovarian
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FIG. 1. Immunocytochemical staining of ovarian tumors with antibodies to cyclin D1, c-erb-B2, and estrogen receptor. (a) Surface epithelial cells (arrows) of the normal ovary fail to stain with antiserum to cyclin D1. (b) Epithelial cells in a mucinous cystadenoma fail to stain with antiserum to cyclin D1. (c) Epithelial cells in a well-differentiated mucinous carcinoma show strong nuclear staining with antiserum to cyclin D1. The stromal component of the tumor fails to stain. (d) A well-differentiated endometrioid tumor showing variable nuclear staining with cyclin D1 antiserum. (e) Endometrioid carcinoma showing strong nuclear staining with antiserum to estrogen receptor. (f) Serous papillary adenocarcinoma showing moderate uniform expression of estrogen receptor. (g) A serous papillary carcinoma different from that shown in (f) fails to stain with antiserum to estrogen receptor. (h) A poorly differentiated carcinoma showing weak membrane staining with antiserum to c-erb-B2. (i) Endometrioid carcinoma showing strong membrane staining with c-erb-B2 antiserum. Note that the stromal component of the tumor (center) fails to stain. (j) The same well-differentiated mucinous tumor as shown in (c) also stains strongly with antiserum to c-erb-B2. Again, only the epithelial cells stain. Magnification of all photographs, 140.
tumors. This number is roughly comparable to the number of tumors overexpressing the c-erb-B2 oncogene both in our study (23%) and in the 20 to 30% reported in other studies [29–31]; however, the spectra of tumors overexpressing these two oncoproteins are very different. With one excep-
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tion, tumors expressing cyclin D1 do not express c-erb-B2. This exception was a well-differentiated mucinous tumor. This suggests that, in general, cyclin D1 is overexpressed in a subset of ovarian tumors different from those that overexpress the c-erb-B2 oncogene.
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FIG. 1—Continued
In our study a correlation was found between tumor grade and cyclin D1 overexpression. Cyclin D1 expression is more commonly found in borderline tumors and grade 1 carcinomas than in grade 2 and 3 carcinomas. There are a number of possible interpretations of this result. A higher frequency of cyclin D1 overexpression in borderline and lower-grade tumors suggests that deregulation of cyclin D1 expression may be a relatively early event in the pathogenesis of epitheTABLE 3 Relationship between Tumor Grade and Expression of Cyclin D1, c-erb-B2, and Estrogen Receptors Tumor grade
Cyclin D1 positivea
ER positiveb
c-erb-B2 positivec
2/3
0/3
0/3
5/18 3/10
4/18 2/10
3/10 0/2
3/10 1/2
Borderline
8/21d Grade 1 Grade 2
6/18 1/10
Grade 3 Mixed
1/10 1/2
lial ovarian cancer. In contrast, overexpression of the c-erbB2 gene may generally be a later event; a correlation with poor prognosis has been associated with overexpression of this oncogene in some studies [29–31]; however, it is possible that many of the borderline/low-grade, cyclin D1-overexpressing tumors are distinctly different lesions that have a pathogenesis different from that of many higher-grade tumors. Other types of mutation that result in higher-grade tumors may be underrepresented in borderline/lower-grade tumors. Further studies in which parameters such as stage and survival are included are clearly necessary to gain a better understanding of the role that cyclin D1 plays in ovarian cancer and its possible use as a prognostic indicator. TABLE 4 Relationship between Tumor Histology and Expression of Cyclin D1, c-erb-B2, and Estrogen Receptors
2/20d
a
Tumors with ú10% positive nuclei. Tumors with ú50% positive nuclei. c Tumors with some or all cells showing definite membrane staining. d Significantly more tumors borderline/Grade 1 cyclin D1 positive than grade 2/grade 3 cyclin D1 positive (P Å 0.035, Fisher exact test). b
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Tumor histology
Cyclin D1 positive
c-erb-B2 positive
ER positive
Serous Mucinous Endometrioid Clear cell Poorly differentiated
1/8 3/7 3/14 2/5 1/4
2/8 3/7 2/14 1/5 2/4
3/8 0/7 5/14 0/5 1/4
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TABLE 5 Analysis of Combinations of Gene Products in Ovarian Tumors Combination Cyclin Cyclin Cyclin Cyclin
Number of tumors
D1//c-erb-B2/ D1//c-erb-B20 D10/c-erb-B2/ D10/c-erb-B20
1/43 10/43 9/43 23/43 P Å 0.162
Cyclin Cyclin Cyclin Cyclin
D1//ER/ D1//ER0 D10/ER/ D10/ER0
2/43 9/43 9/43 23/43 P Å 0.269
c-erb-B2//ER/ c-erb-B2//ER0 c-erb-B20/ER/ c-erb-B20/ER0
0/43 10/43 11/43 22/43 P Å 0.034
Furthermore, the development of appropriate animal models for epithelial ovarian cancer will allow more revealing functional studies to be carried out to determine the phenotypic consequences of cyclin D1 overexpression in ovarian epithelium. Cyclin D1 has been shown to enhance amplification of the CAD gene by more than 1000-fold in rat liver epithelial cells, apparently due to loss of control of the G1 –S cell cycle checkpoint [8]. Although c-erb-B2 is frequently amplified and overexpressed in ovarian cancers, its expression does not appear to be deregulated by cyclin D1 expression. In our study, c-erb-B2 expression did not correlate with tumor grade but did correlate with absence of estrogen receptors. The frequency of ovarian tumors showing overexpression of cyclin D1 in our study is rather lower than the reported frequencies of breast and certain other cancers [17, 32] showing overexpression of this protein. It is only possible to speculate why this may be the case. First, there are almost certainly multiple pathways to tumor development and cyclin D1 overexpression may contribute to only one or a proportion of these pathways. Second, it is probable that the cyclin D1 protein is functionally redundant and that other proteins can substitute for its contribution to a carcinogenic pathway. Cancer cells may use other mechanisms for cell cycle progression from G1 to S phase. For example, mutation or downregulation of expression of the multiple tumor suppressor gene (MTS1) may substitute for cyclin D1 overexpression. Moreover, an absence of a requirement for cyclin D1 has been shown in breast cancer cell lines with defects in Rb function [33]. This having been said, genetic changes to MTS1 appear to be infrequent in ovarian cancer [34, 35]; similarly genetic changes to the Rb gene and abnormal Rb gene expression have also been reported to be uncommon
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in ovarian cancer [36–38]. Whatever the explanation, it is clear that cyclin D1 overexpression is not an obligatory requirement for the proliferation of epithelial ovarian cancers. There has been some suggestion that deregulation of cyclin D1 expression is also an early event in the pathogenesis of breast cancer. When a comparison was made between matched lesions at different stages of tumor progression, cyclin D1 mRNA expression was found to be an early event that is maintained throughout progression including metastatic spread [22]. This suggests that cyclin D1-expressing breast cancers need to retain expression of this gene product to develop into a fully invasive, metastatic cancer. It would be interesting to determine whether this is the case in epithelial ovarian cancer. In breast cancer cells, cyclin D1 expression has been shown to be induced by steroids and its expression inhibited by antiestrogens. Therefore, we investigated whether overexpression of cyclin D1 in ovarian tumors required the presence of estrogen receptors. In our study, we found no correlation between cyclin D1 expression and the presence or absence of estrogen receptors. Clearly a level of estrogen receptor expression detectable by immunocytochemistry is not a requirement for deregulated cyclin D1 expression in ovarian cancers. In conclusion, cyclin D1 overexpression is found in approximately one-quarter of ovarian cancers. Overexpression of this gene occurs predominantly in tumors that do not overexpress the c-erb-B2 oncogene, but does not correlate with the presence of estrogen receptors. The functional consequences of deregulated cyclin D1 expression in ovarian epithelium and its relationship to patient prognosis deserve further investigation. ACKNOWLEDGMENTS The authors thank Dr. Hilary Buckley (St. Mary’s Hospital, Manchester, UK) and Dr. M. C. Anderson (Queens Medical Centre, University of Nottingham Medical School) for providing some of the tumor samples and pathology reports. This work was funded by WellBeing.
REFERENCES 1. Draetta G: Mammalian G1 cyclins. Curr Opin Cell Biol 6:8429–8460, 1994 2. Baldin V, Lukas J, Marcote MJ, Pagano M, Draetta G: Cyclin D1 is a nuclear protein required for cell cycle progression in G1 . Genes Dev 7:812–821, 1993 3. Quelle D, Ashmun R, Shurtleff S, Kato J, Bar-Sagi D, Roussel M, Sherr C: Overexpression of mouse D-type cyclins accelerates G1 phase in rodent fibroblasts. Genes Dev 7:1559–1571, 1993 4. Lukas J, Pagano M, Staskova Z, Draetta G, Bartek J: Cyclin D1 protein oscillates and is essential for cell cycle progression in human tumor cell lines. Oncogene 9:707–718, 1994 5. Jiang W, Khan SM, Zhou P, Zhang YJ, Cacace AM, Infante AS: Overexpression of cyclin D1 in rat fibroblasts causes abnormalities in
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CYCLIN D1 OVEREXPRESSION IN OVARIAN CANCER growth control, cell cycle progression and gene expression. Oncogene 8:3447–3457, 1993 6. Hartwell L: Defects in cell cycle checkpoints may be responsible for the genomic instability of cancer cells. Cell 71:543–546, 1992 7. Almason A, Linke SP, Paulson TG, Wall GM: Genetic instability as a consequence of inappropriate entry into the cell cycle progression through S phase. Cancer Metast Rev 14:59–73, 1995 8. Zhou P, Jiang W, Weghorst CM, Weinstein IB: Overexpression of cyclin D1 enhances gene amplification. Cancer Res 56:36–39, 1996 9. Sutherland RL, Hamilton JA, Sweeney KJE, Watts CKW, Musgrove EA: Expression and regulation of cyclin genes in breast cancer. Acta Oncol 34:651–656, 1995 10. Arnold A, Kim HG, Gaz RD, Eddy RL, Fukusima Y, Kronenberg HM: Molecular cloning and chromosomal mapping of DNA rearranged with the parathyroid hormone gene in a parathyroid adenoma. J Clin Invest 83:2034–2040, 1989 11. Williams ME, Swerdlow SH, Rosenberg CL, Arnold A: Centrocytic lymphoma: A B-cell non-Hodgkin’s lymphoma characterised by chromosome 11 bcl-1 and PRAD1 rearrangements. Curr Top Microbiol Immunol 182:325–329, 1992
23.
24.
25.
26.
27.
28.
12. Brookes S, Lammie GA, Schuuring E, Dickson C, Peters G: Linkage map of a region of human chromosome band 11q13 amplified in breast and squamous cell tumors. Genes Chromosom Cancer 4:290–301, 1992
29.
13. Jiang W, Kahn SM, Tomita N, Zhang YJ, Lu SH, Weinstein IB: Amplification and expression of the human cyclin D1 gene in oesophageal cancer. Cancer Res 52:2980–2983, 1992
30.
14. Lammie GA, Peters G: Chromosome 11q13 abnormalities in human cancer. Cancer Cells 3:413–419, 1991 15. Champeme MH, Bieche I, Lizard S, Liderau R: 11q13 amplification in local recurrence of human primary breast cancer. Genes Chromosom Cancer 12:128–133, 1995
31.
16. Buckley MF, Sweeney KJE, Hamilton JA, Sini RL, Manning DL, Nicholson RI, deFaxio A, Watts CKW, Musgrove EA, Sutherland RL: Expression and amplification of cyclin genes in human breast cancer. Oncogene 8:2127–2133, 1993
32.
17. Gillet C, Frantl V, Smith R, Fisher C, Bartek J, Dickson C, Barnes D, Peters G: Amplification and overexpression of cyclin D1 in breast cancer detected by immunocytochemical staining. Cancer Res 54:1812–1817, 1994 18. Bartkova J, Lukas J, Muller H, Lutzhoft D, Strauss M, Bartek J: Cyclin D1 protein expression and function in human breast cancer. Int J Cancer 57:353–361, 1994
33.
34.
35.
19. Worsley SD, Jennings BA, Khalil KH, Mole M, Girling AC: Cyclin D1 amplification and expression in human breast carcinoma: Correlation with histological prognostic markers and oestrogen receptor expression. J Clin Pathol 49:M46–M50, 1996
36.
20. Michalides R, Hageman P, van Tinteren H, Houben L, Weintjens E, Klompmaker R, Pterse J: A clinicopathological study on overexpression of cyclin D1 and of p53 in a series of 248 patients with operable breast cancer. Br J Cancer 73:728–734, 1996
37.
21. Fantl V, Richards MA, Smith R: Gene amplification on chromosome band 11q13 and estrogen receptor status in breast cancer. Eur J Cancer 26:423–429, 1990 22. Weinstat-Saslow D, Merlino MJ, Manrow RE, Lawrence JA, Bluth RF, Wittenbel KD, Simpson JF, Page DL, Steeg PS: Overexpression of
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38.
195
cyclin D mRNA distinguishes in situ and invasive breast carcinomas from non-malignant lesions. Nature Med 1:1256–1260, 1996 Wang TC, Cardiff RD, Zukerberg L, Lees E, Arnold A, Schmidt EV: Mammary hyperplasia and carcinoma in MMTV–cyclin D1 transgenic mice. Nature 369:669–671, 1994 Bodrug SE, Warner BJ, Bath ML, Lindeman GJ, Harris AW, Adams JM: Cyclin D1 transgene impedes lymphocyte maturation and collaborates in lymphoma genesis with the myc gene. EMBO J 13:2124–2130, 1994 Campbell IG, Jones TA, Foulkes WD, Trowsdale J: Folate binding protein is a marker for ovarian cancer. Cancer Res 51:5329–5338, 1991 Hsu SM, Raine L, Fanger H: Use of avidin–biotin–peroxidase complex (ABC) in immunoperoxidase techniques. J Histochem Cytochem 29:577, 1981 Norton AJ, Jordan S, Yeomans P: Brief, high temperature heat denaturation (pressure cooking): A simple and effective method of antigen retrieval for routinely processed tissues. J Clin Pathol 173:371–379, 1994 Berchuck A, Kamel A, Whitaker R, Kerns B, Olt G, Kinney R, Soper T, Dodge R, Clarke-Pearson P, McKenzie S, Yin S, Bast RC: Overexpression of HER-2/neu is associated with poor survival in advanced epithelial ovarian cancer. Cancer Res 50:4087–4091, 1990 Slamon DJ, Godolphin W, Jones LA, et al.: Studies of HER-2/neu proto-oncogene in human breast and ovarian cancer. Science 244:707– 712, 1989 Felip E, Del Campo JM, Rubio D, Vidal MT, Colomer R, Bermejo B: Overexpression of c-erb-B2 in epithelial ovarian cancer. Cancer 75:2147–2152, 1995 Zhang S-Y, Caamano J, Cooper F, Guo X, Klein-Szanto AJP: Immunohistochemistry of cyclin D1 in human breast cancer. Am J Clin Pathol 102:695–698, 1994 Zhang S-Y, Caamano J, Cooper F, Guo X, Klein-Szanto AJP: Immunohistochemistry of cyclin D1 in human breast cancer. Am J Clin Pathol 102:695–698, 1994 Lukas J, Bartkova J, Rohde M, Strauss M, Bartek J: Cyclin D1 is dispensible for G1 control in retinoblastoma gene-deficient cells independent of cdk4 activity. Mol Cell Biol 15:2600–2611, 1995 Rodabaugh KJ, Biggs RB, Qureshi JA, Barrett AJ, Welch WR, Bell DA, Berkowitz RS, Mok SC: Detailed deletion mapping of chromosome 9p and p16 gene alterations in human borderline and invasive epithelial ovarian tumors. Oncogene 11:1249–1254, 1995 Campbell IG, Beynon G, Davis M, Englefield P: LOH and mutation analysis of CDKN2 in primary human ovarian cancers. Int J Cancer 63:222–225, 1995 Dodson MK, Cliby WA, Xu HJ, DeLacey KA, Hu SX, Keeney GL, Li J, Podratz KC, Jenkins RB, Benedict WF: Evidence of functional Rb protein in epithelial ovarian carcinomas despite loss of heterozygosity at the Rb locus. Cancer Res 54:610–613, 1994 Kim TM, Benedict WF, Xu HJ, Hu SX, Gosewehr J, Velicescu M, Yin E, Zheng J, D’Ablaing G, Dubeau L: Loss of heterozygosity on chromosome 13 is common only in the biologically more aggressive subtypes of ovarian epithelial tumors and is associated with normal retinoblastoma gene expression. Cancer Res 54:605–609, 1994 Liu Y, Heyman M, Wang Y, Falkmer IJ, Hising C, Szekely L, Einhorn S: Molecular analysis of the retinoblastoma gene in primary ovarian cancer cells. Int J Cancer 58:663–667, 1994
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