ANTICANCER RESEARCH 32: 3993-4000 (2012)

The Expression of β-Microseminoprotein but Not CRISP3 Is Reduced in Ovarian Cancer and Correlates to Survival

RUDI HENRIKSEN1, ÅKE LUNDWALL2, LENE UDBY3 and PER FERNLUND2

Departments of 1Obstetrics and Gynaecology and 2Clinical Chemistry, University Hospital SUS, Malmö, Sweden; 3Department of Hematology, Granulocyte Research Laboratory, Copenhagen University Hospital, Rigshospitalet, Copenhagen, Denmark

Abstract. Background: β−Microseminoprotein (MSMB) is that decreased expression was not observed in invasive tumor an abundant in seminal plasma. Most of it is present cells. Conclusion: MSMB and CRISP3 were widely as a free protein but a small part is bound to cysteine-rich distributed in ovaries and in ovarian tumors; the expression secretory protein 3 (CRISP3) as a non-covalent complex. of MSMB fits well with a tumor-suppressor function in Even though their physiological function is unknown, both ovarian carcinogenesis. MSMB and CRISP3 have been ascribed roles in prostate carcinogenesis. Thus, several recent experimental studies Ovarian cancer has the highest mortality rate of all types of indicate a tumor-suppressor role for MSMB. The present cancers of the female reproductive system. In spite of study was undertaken in order to evaluate, for the first time, advances in cytoreductive surgery and chemotherapy, the the expression of MSMB and CRISP3 in ovaries and in overall 5-year survival has only marginally increased (from ovarian tumors and to determine if their expression might 37% to 45%) during the last three decades (1). Surgery has indicate a role in ovarian tumor development. Materials and become more centralized and specialized and several drugs Methods: Biopsies from prospectively collected samples from in almost any combination have been used in numerous ovaries and benign, borderline and invasive ovarian tumors studies. Despite this, according to a recent conference report, were analyzed for expression of MSMB and CRISP3 by progress in survival seems to have stopped, which makes immunohistochemistry. In patients with ovarian cancer the development of new strategies necessary (2). To achieve this, expression was compared to survival. Results: Both MSMB a detailed knowledge on tissue biology during ovarian and CRISP3 were strongly stained in ovarian epithelial cells carcinogenesis is important. However, although reports (3, 4) and weakly stained in the stroma. In ovarian blood vessels, have described the possible contribution of several growth- CRISP3 exhibited strong to medium staining, while MSMB regulatory factors, a complete model of the molecular was only weakly expressed. In benign and borderline tumors changes that occur during tumor development still seems far the staining pattern was similar to the one observed in the away. MSMB, also known as prostatic secretory protein, of ovaries. In invasive neoplasms, the expression of MSMB in 94 amino acids is one of the three major secreted by the tumor cells was significantly reduced. In univariate the prostate gland (5). Remarkably, the functions of MSMB analysis, decreased expression of MSMB correlated to in the gland and in sperm are still unknown. MSMB is reduced survival. No correlation was found with stage, the produced mainly by the prostate gland but has also been strongest prognostic indicator for ovarian cancer, which found in other tissues (6, 7). MSMB mRNA has been supports an independent role of MSMB in ovarian described in the ovaries (8), but to our knowledge, no further carcinogenesis. For CRISP3, a staining pattern comparable studies concerning its expression or its possible role in to that for MSMB was observed in all groups, except the fact ovarian physiology or pathology have been published. CRISP3 was first isolated from neutrophilic leukocytes (9, 10), but is also found in exocrine secretions such as saliva, pancreatic fluid and seminal plasma. In seminal plasma, it is Correspondence to: Rudi Henriksen, MD, Ph.D., Department of present as a non-covalent complex with MSMB (11). Like Obstetrics and Gynaecology, University Hospital, 20502 Malmö, MSMB, the physiological function of CRISP3 is unknown, Sweden. Tel: +46 40332168, Fax: +46 40962600, e-mail: [email protected] as is the significance of the binding between MSMB and CRISP3. Patients with positive for CRISP3 Key Words: β-Microseminoprotein, CRISP3, immunohistochemistry, in the tumor tissue were less likely to remain recurrence-free ovary, ovarian cancer, survival. (12). In the current work, we studied the expression of

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MSMB and CRISP3 with immunohistochemistry in ovaries neoplasms, tumor cells in 25 out of 37 biopsies exhibited a and in benign and malignant ovarian tumors and correlated weak to intermediate staining, which deviated significantly the presence of these proteins with tumor development. from the staining of ovaries (p=0.01) and benign (p=0.005) and borderline tumors (p=0.05) (Table II). Examples of the Materials and Methods staining in various samples are shown in Figure 1. Survival related to staining was estimated by dividing the material Patient samples. Fresh surgical specimens were collected into three groups, strong staining (9 out of 12 patients with prospectively and frozen immediately, and kept at –70˚C until available survival data), intermediate staining (10 patients) analyzed. The patient material was anonymized according to the and weak or absent staining (15 patients). A Kaplan Meier guidelines of the Malmö-Lund Ethical Committee. Biopsies were curve showed that decreased survival correlated to decreased obtained from 8 normal ovaries, 13 benign, 5 borderline tumors and 37 invasive epithelial ovarian carcinomas. There were 12 in stage I, staining, with the shortest survival in the group with most 4 in stage II, 16 in stage III, 2 in stage IV and for 3 patients the reduced staining and intermediate survival in the group stage was unknown. None of the patients with cancer had been having intermediate staining. A log rank test confirmed the subject to treatment prior to surgery. Survival data were obtained significant difference between the groups having high and for all patients except three. low expression (p=0.02) (Figure 3). In the stroma and blood vessels, the expression of MSMB was generally weak or Antibodies and immunohistochemistry. The rabbit antisera raised absent, presenting no difference between the groups. against human MSMB and the aminoterminal part of recombinant Immunostaining for CRISP3 was strong in ovarian epithelial human CRISP3 has been described previously (7, 9). cells, as well as in benign and malignant tumor cells, with a Immunostaining was performed as described previously (13), with minor modifications. Six-micrometer-thick cryosectioned tissue minor non-significant reduction in the invasive neoplasms sections fixed in 4% paraformaldehyde were treated for 30 min with (Table I and II, Figure 2). In the stromal part, weak or absent 0.3% hydrogen peroxide to block endogenous peroxidase activity. staining was observed. In the blood vessels, strong staining Endogenous avidin-binding activity was blocked by sequential was seen in the ovaries, but staining was weak or absent in incubation with avidin and biotin using a blocking kit (Vector the tumor groups. Laboratories, Burlingame, CA, USA). Non-specific binding of the We found no correlation for neither MSMB nor CRISP3 secondary antibody was blocked by incubation with normal goat expression to stage or to earlier published expression of p53 serum diluted 1:10 with phosphate buffered saline. After incubation with the primary antibody (diluted 1:1000) overnight at 4˚C in a (17), Ki67, S-phase fraction or DNA ploidy (18) (results not humidified chamber, the tissue sections were incubated with shown). biotinylated goat-anti rabbit immunoglobulin (Vector Laboratories). The immunoreaction was visualized with a Vectastain Elite ABC Discussion complex (Vector Laboratories) using 0.02% hydrogen peroxide as a substrate and 3,3’-diaminobenzidine tetrahydrochloride as a Our study on the expression of MSMB and CRISP3 in chromogen (Dako, Glostrup, Denmark). Finally the sections were normal and malignant ovarian tissue contributes several new briefly counterstained for 15 sec in Meyer’s hematoxylin. The staining was estimated as being strong if more than two thirds of observations. Thus, both proteins were for the first time the cells were immunoreactive, intermediate if one third to two described in ovaries as well as in ovarian tumors. MSMB was thirds stained positively and weak if less than one third of the cells primarily localized to epithelial cells and benign tumor cells were positively stained. with only a weaker or absent staining in stroma and in blood vessels. Furthermore, MSMB immunoreactivity was equally Statistical methods. Differences in protein expression between the strong in borderline tumors cells. However, in invasive cancer groups were estimated with the two-tailed Fisher’s exact probability cells, a significantly reduced expression was observed which test (14). The survival times were assessed from the day of surgery. Cumulative survival curves were constructed according to the was correlated to a reduced survival. Although the analysis Kaplan Meier method (15) and differences in survival were was univariate, the significance is supported by the observed estimated with the log-rank test (16). lack of correlation of MSMB to stage, the strongest prognostic parameter in ovarian cancer. Neither was the Results expression correlated to other prognostic factors such as cell proliferation and ploidy. Thus, when the malignant cells MSMB was strongly stained in ovarian epithelium in all acquired invasive potential, MSMB expression decreased or eight samples (Table I). Medium and weak staining was did not appear the cells. Although not a proof, this pattern fits observed in the stroma in four cases each and in one case well with a suppressor function of MSMB and is in line with each for blood vessels. In 9 out of 10 benign ovarian tumors, recent observations in prostate cancer, as described below. MSMB was strongly expressed in tumor cells while one Initially, MSMB was described as one of three major exhibited intermediate staining. Tumor cells from five proteins in seminal plasma with an unknown biological role. borderline tumors all exhibited strong expression. In invasive Later it was found in other tissues, including genital organs.

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Table I. Number of patients with tumors exhibiting strong, intermediate Table II. Expression of β-Microseminoprotein (MSMB) and cysteine- or weak to absent expression of β-Microseminoprotein (MSMB) and rich secretory protein 3 (Crisp3) in epithelial cells and tumor cells. The cysteine-rich secretory protein 3 (Crisp3) in different compartments of staining was classified as strong, intermediate or weak to absent, as ovaries and ovarian tumors, as described in the Materials and Methods. described in the Materials and Methods. In the table, only patients with tumor exhibiting high or low expression are compared. Strong/intermediate/weak staining Protein Expression MSMB CRISP3 MSMB Strong Weak Ovary Ovary 8 0 Epithelium 8/0/0 4/0/0 Benign 9 0 Stroma 0/4/4 1/3/0 Borderline 5 0 Blood vessels 0/1/1 3/1/0 Malignant 12 15 Benign tumors Tumor cells 9/1/0 12/1/0 Mal vs. ovary p=0.01 Stroma 0/0/10 0/9/4 Mal vs. benign p=0.005 Blood vessels 0/0/10 0/5/8 Mal vs. borderline p=0.05 Borderline tumors Tumor cells 5/0/0 3/1/0 Crisp3 Strong Weak Stroma 0/0/5 0/2/2 Ovary 4 0 Blood vessels 0/0/5 0/2/2 Benign 12 0 Malignant tumors Borderline 3 0 Tumor cells 12/10/15 27/7/0 Malignant 27 0 Stroma 0/3/34 1/17/16 Blood vessels 0/0/37 0/11/23 No significant differences were observed between any of the groups.

In recent years, MSMB has been coupled to the development role in ovarian carcinogenesis. The strong expression in of prostate cancer. Thus, several publications have shown ovarian epithelium also indicates a role in ovarian decreased expression of MSMB in prostate cancer cells physiology. Ovarian epithelium is a dynamic structure with a compared to normal prostate (19-25). Decreased expression determinant role in healing and scar formation after monthly has been reported to correlate to shorter recurrence-free time repeated ovulations. Whether MSMB may play a role in this (12) and lower survival (26), although contrary observations process of repeated proliferative activity is unknown but in have also been reported (27, 28). Furthermore, low light of the proposed involvement in ovarian tumor concentration of MSMB in serum correlated to a higher risk development, this aspect deserves further study. of having prostate cancer detected at biopsy (29). The biology of benign ovarian tumors at the molecular Beside the pattern of expression, experimental data level is not well-known. Using the proliferation marker Ki- support a tumor suppressor-like role of MSMB in prostate 67, we found no signs of cell proliferation (18). The route carcinogenesis. In animal models, naturally-occurring from ovarian epithelium to invasive epithelial tumor is still MSMB (30, 31) as well as recombinant MSMB (32) unknown and thus, it is an open question as to how many demonstrated an ability to reduce tumor growth. malignant tumors develop through benign and borderline Furthermore, a Canadian group has studied the effect of the equivalents. The detection of MSMB in benign tumor cells amino acid 31-45 region of MSMB (PCH3145), which was is new and its functional significance unknown. found sufficient to elicit MSMB-mediated antitumor effects What regulates the expression of MSMB is not known and (33). Studies in a rat model, as well as in humans, have thus, the reason for the decreased expression of MSMB revealed the effects of MSMB on several parameters, with correlated to malignancy is not understood. However, recent potential tumor-suppressing effects, including vascular observations in prostate cancer point to a possible explanation endothelial growth factor (34), matrix metalloproteinase-9 which may fit with the decreased expression in malignant (35, 36) and the cell-surface glycoprotein CD44 (35). In vivo ovarian neoplasms. The loss of expression of MSMB in studies of rats inoculated with rat prostate cancer Mat Ly Lu hormone-refractory prostate cancer correlated with an cells showed that treatment with PCH3145 resulted in a increased expression of the polycomb protein EZH2 (37). The dose-dependent decrease in tumor volume and a delay in transcription of the coding for MSMB was found to be development of skeletal metastases (33). Thus, solid clinical repressed by EZH2 via trimethylation of histone H3 on Lys27 and experimental data indicate a protective role of MSMB in in androgen-refractory but not in androgen-sensitive prostate prostate carcinogenesis. Our observations on the protein cancer, indicating a causal relation (37). Strong expression of expression in ovarian tumors are new and support a similar EZH2 is associated with aggressive tumor subgroups in other

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Figure 1. Immunohistochemical expression of β-Microseminoprotein (MSMB) in ovaries and ovarian tumors. a: Ovary showing strong expression in the epithelium; b: benign mucinous tumor with strong staining of the benign tumor cells; c: benign Brenner tumor and strong staining of the epithelial benign tumor cells; d: borderline serous tumour showing strong expression of the tumor cells; e: weak expression in endometrioid cancer (lower part) and in ovarian stroma (upper part), while the epithelial cells stain positively; f: seropapillary cancer with weakly stained tumor cells. Magnification ×200 except in e: ×100.

Figure 2. Immunohistochemical expression of cysteine-rich secretory protein 3 (Crisp3) in ovaries and ovarian tumors. a: Ovary with strongly- stained epithelium; b: ovarian stroma and positively stained blood vessels; c: benign mucinous tumor with strong expression in tumor cells; d: seropapillary cancer showing strong staining of tumor cells. Magnification ×200 in all cases except in d) with magnification ×100.

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In our present work the localizations of the staining for MSMB and CRISP3 were generally overlapping and thus leave the possibility open for interactions as has been described in the prostate. However, as no significant changes in staining of CRISP3 were observed in the malignant tumor cells compared to epithelial or benign tumor cells, our findings do not seem to support a role for CRISP3 in ovarian tumor development. In conclusion, we have, for the first time, described the existence of MSMB and CRISP3 in the ovaries and in ovarian tumors. In patients with invasive tumors, univariate survival analysis showed a correlation between decreased expression and decreased survival. Our results direct to a tumor-suppressor function of MSMB in ovarian Figure 3. Survival correlated to expression of β-Microseminoprotein carcinogenesis, corresponding to recently published (MSMB) in ovarian cancer. Survival in groups of patients having strong, experimental data for prostate cancer. Therefore, further intermediate or weak expression of MSMB in ovarian cancer cells. research in ovarian cancer is imperative to reveal if the Survival was significantly longer in patients with high expression MSMB gene product or a fragment of it may allow new compared to the group with weak expression (p=0.02). No difference options in the treatment of patients with ovarian cancer. was observed between high and intermediate groups nor between groups with intermediate and low expression (p=0.32 for both). Acknowledgements

Professor Pirkko Härkönen, Department of Clinical Research tissues (38). In analysis of blood vessels from Center, Lunds University, Malmö, Sweden is deeply acknowledged ovaries and ovarian cancer using microarrays, overexpression for support with laboratory facilities and material. The Authors of EZH2 in tumor-associated blood vessels was observed (39) declare that they have no conflict of interest regarding this study. and, recently, increased expression of EZH2 was found in tumor cells, as well as in tumor vasculature, and was References associated with reduced survival in patients with ovarian cancer (40). We found weak to intermediate expression of 1 Jemal A, Siegel R, Ward E, Murray T, Xu J, Smigal C and Thun MSMB in blood vessels in two of eight ovaries, but no MJ: Cancer statistics, 2006. CA Cancer J Clin 56: 106-130, corresponding staining in the tumors. Whether the previously 2006. described overexpression of EZH2 in ovarian cancer cells and 2 Ashworth A, Balkwill F, Bast RC, Berek JS, Kaye A, Boyd JA, Mills G, Weinstein JN, Woolley K and Workman P: blood vessels may contribute to the decreased expression of Opportunities and challenges in ovarian cancer research, a MSMB in ovarian cancer is unknown. Another interesting perspective from the 11th Ovarian Cancer Action/HHMT Forum, possible regulatory mechanism is the recent observation that Lake Como, March 2007. Gynecol Oncol 108: 652-657, 2008. EZH2 is suppressed by activated p53 (41). p53 is functionally 3 Wong AS and Leung PC: Role of endocrine and growth factors inactivated in about 50% of malignant neoplasms including on the ovarian surface epithelium. J Obstet Gynaecol Res 33: 3- malignant ovarian neoplasms, as in our own material (17). 16, 2007. Increased EZH2, mediated by decreased p53 activity, may be 4 Yoshida S, Furukawa N, Haruta S, Tanase Y, Kanayama S, a new regulatory mechanism of importance in physiology, as Noguchi T, Sakata M, Yamada Y, Oi H and Kobayashi H: Expression profiles of involved in poor prognosis of well as in carcinogenesis. However, the exact significance of epithelial ovarian carcinoma: a review. Int J Gynecol Cancer 19: p53 in the regulation of EZH2 must be further studied. 992-997, 2009. The expression of CRISP3 has been shown to be highly up- 5 Abrahamsson PA and Lilja H: Three predominant prostatic regulated in prostate cancer (42, 43) and associated with proteins. Andrologia 22(Suppl 1): 122-131, 1990. recurrence after radical prostatectomy (12). In ovaries and 6 Ulvsback M, Lindstrom C, Weiber H, Abrahamsson PA, Lilja H ovarian tumors, we found-strong expression in ovarian and Lundwall A: Molecular cloning of a small prostate protein, epithelium, as well as in benign tumor cells, and a minor known as beta-microsemenoprotein, MSMB or beta-inhibin, and reduction of staining in malignant tumor cells. Interestingly, the demonstration of transcripts in non-genital tissues. Biochem Biophys Res Commun 164: 1310-1315, 1989. blood vessels in the ovaries were also strongly stained. There 7 Weiber H, Andersson C, Murne A, Rannevik G, Lindstrom C, was comparatively moderate- to weak-staining in half of the Lilja H and Fernlund P: Beta- microseminoprotein is not a tumors. This is the first association of CRISP3 with blood prostate-specific protein. Its identification in mucous glands and vessels and again its significance is completely unknown. secretions. Am J Pathol 137: 593-603, 1990.

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36 Annabi B, Bouzeghrane M, Currie JC, Dulude H, Daigneault L, 41 Tang X, Milyavsky M, Shats I, Erez N, Goldfinger N and Rotter Garde S, Rabbani SA, Panchal C, Wu JJ and Beliveau R: V: Activated p53 suppresses the histone methyltransferase EZH2 Inhibition of MMP-9 secretion by the anti-metastatic MSMB- gene. Oncogene 23: 5759-2769, 2004. derived peptide PCK3145 requires cell surface laminin receptor 42 Kosari F, Asmann YW, Cheville JC and Vasmatzis G: Cysteine- signaling. Anticancer Drugs 17: 429-438, 2006. rich secretory protein-3: a potential biomarker for prostate 37 Beke L, Nuytten M, Van Eynde A, Beullens M and Bollen M: cancer. Cancer Epidemiol Biomarkers Prev 11: 1419-1426, 2002. The gene encoding the prostatic tumor suppressor MSMB is a 43 Bjartell A, Johansson R, Björk T, Gadaleanu V, Lundwall A, target for repression by the polycomb group protein EZH2. Lilja H, Kjeldsen L and Udby L: Immunohistochemical Oncogene 26: 4590-4595, 2007. detection of cysteine-rich secretory protein 3 in tissue and in 38 Bachmann IM, Halvorsen OJ, Collett K, Stefansson IM, Straume serum from men with cancer or benign enlargement of the O, Haukaas SA, Salvesen HB, Otte AP and Akslen LA: EZH2 prostate gland. Prostate 66: 591-603, 2006. expression is associated with high proliferation rate and aggressive tumor subgroups in cutaneous melanoma and cancers of the endometrium, prostate, and breast. J Clin Oncol 24: 268-273, 2006. 39 Lu C, Bonome T, Li Y, Kamat AA, Han LY, Schmandt R, Coleman RL, Gershenson DM, Jaffe RB, Birrer MJ and Sood AK: Gene alterations identified by expression profiling in tumor- associated endothelial cells from invasive ovarian carcinoma. Cancer Res 67: 1757-1768, 2007. Received June 12, 2012 40 Chunhua L, Hee Dong H and Lingegowda SM: Regulation of Revised July 2, 2012 tumor angiogenesis by EZH2. Cancer Cell 18: 185-197, 2010. Accepted July 3, 2012

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