Insulin-like Growth Factor Binding Proteins IGFBP 3 , IGFBP 4 , and IGFBP 5 Predict Endocrine Responsiveness in Patients with Ovarian Cancer

Purpose: This study sought to explore the predictive value of the insulin-like growth factor (IGF) binding proteins (IGFBP) as markers of response in ovarian cancer patients treated with the aromatase inhibitor letrozole. Experimental Design: IGFBP mRNA expression in cell lines was measured by quantitative reverse transcription-PCR and IGFBP protein expression measured in sections from primary tumors of patients treated with letrozole by semiquantitative immunohistochemistry. Results:Quantitative reverse transcription-PCR analysis showed that IGFBP3 and IGFBP5 were down-regulated and IGFBP4 was up-regulated by 17h-estradiol (E2) in an estrogen receptor (ER)^ positive ovarian cancer cell line. Expressions of IGFBP1, IGFBP2, and IGFBP6 were unaffected by E2. The E2 modulation of these genes was reversed by tamoxifen. Using ERa-specific (propyl pyrazole triol) and ERh-specific (diarylpropionitrile) agonists, the gene expressionmodulations produced by E2 could be replicated by propyl pyrazole triol but not by diarylpropionitrile. For ovarian cancer patients being treated with letrozole, we tested the predictive value of the IGFBPs in paraffin-fixed sections from their primary tumors by semiquantitative immunohistochemistry. Using serum CA125 as an indicator of progression/response, significant differences in expression levels of IGFBPs were observed between tumors from CA125 responding/stable patients compared with tumors from progressing patients. Mean immunoscores for IGFBP3 and IGFBP5 were significantly lower, and mean expression of IGFBP4 was significantly higher in tumors from patients demonstrating CA125 response or stabilization compared with CA125 progression. Conclusion: These results indicate that expression levels of certain IGFBP family members in ovarian cancers are estrogen regulated and can, thus, help identify patients who could benefit from endocrine therapy. Epithelial ovarian cancer is the leading cause of death from gynecologic malignancies in the Western world, and new approaches are required to treat this disease. Ovarian cancers frequently express high levels of estrogen receptor a (ERa), which raises the possibility that ERa might represent a therapeutic target. We have previously shown that ERa-positive ovarian cancer cell line models are growth responsive to estrogen and antiestrogen manipulations both in vitro and in vivo (1–4). The antiestrogen tamoxifen has been widely used to treat chemoresistant diseases with inconclusive results (5, 6). More recently, we and others have shown that the aromatase inhibitor letrozole, which acts by depleting levels of estrogen available to ERa, has shown clinical benefit in a subgroup of ovarian cancer patients (7, 8). In a phase II trial of letrozole in patients with ovarian cancer, we showed that a response or stabilization of disease, as defined by CA125 levels, was associated with higher levels of ERa expression, low erbB2 receptor levels, and high epidermal growth factor receptor expression (7), and an extension to this trial has confirmed that expression of ERa predicts sensitivity (9). The overall combined rate of CA125 marker response and stabilization was 32% in the first trial, and the median survival was 14 months with 17% of patients alive at 2 years (7). In the follow-up study that was restricted to patients with moderateto-high ERa-expressing cancers, the CA125 response rate increased to 16% with CA125 stabilization shown in a further 37% of patients (9). To search for predictive markers to help identify estrogensensitive ovarian cancers, we used microarray analysis to highlight a panel of potentially useful gene markers (4). That study explored 17h-estradiol (E2) modulation of gene expression in an ERa-positive ovarian cancer cell line (PE01) in vitro Imaging, Diagnosis, Prognosis Authors’Affiliations: Edinburgh Cancer Research Center and Department of Pathology, University of Edinburgh, Edinburgh, United Kingdom Received 9/12/06; revised12/1/06; accepted12/22/06. 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 with18 U.S.C. Section1734 solely to indicate this fact. Requests for reprints: Simon Langdon, Edinburgh Cancer Research Center, University of Edinburgh, Crewe Road South, Edinburgh, EH4 2XR, United Kingdom. Phone: 44-1317773537; Fax: 44-1317773520; E-mail: simon.langdon@ cancer.org. uk. F2007 American Association for Cancer Research. doi:10.1158/1078-0432.CCR-06-2245 www.aacrjournals. org Clin Cancer Res 2007;13(5) March1, 2007 1438 Research. on April 13, 2017. © 2007 American Association for Cancer clincancerres.aacrjournals. org Downloaded from (4). The gene which showed the most marked change on this 1200–cancer-gene array was insulin-like growth factor (IGF) binding protein (IGFBP) 3, which was down-regulated by estrogen. Because IGFBP4 and IGFBP5 are also well-described estrogen-regulated markers in breast cancer (10–13), we considered that this family might provide valuable predictive information in the context of ovarian cancer. The IGFBP family of binding proteins consists of six structurally related proteins (14, 15). Their role is to bind and regulate the effects of the IGFs, which play major roles in cell proliferation, differentiation, and apoptosis in many cell types. In addition, some of the family members, notably IGFBP3, have direct IGF-independent effects on cellular growth and apoptosis (15). All six family members are expressed in the normal ovary, with IGFBP1-IGFBP5 and IGFBP6 being identified by in situ hybridization and by reverse transcription-PCR, respectively (16–18). These molecules play significant roles in the regulation of both folliculogenesis and steroidogenesis with IGFBP4, in particular, having a major role in the selection of the dominant follicle (16, 19). An association between serum levels of this family and the presence of ovarian cancer has also been identified (20). Although serum levels of IGFBP2 are significantly elevated in patients with ovarian cancer relative to those with benign tumors, expressions of both IGFBP3 and IGFBP5 are lower (20). In this study, we investigated the estrogen regulation of transcription of the IGFBPs in an ER-positive, estrogen-sensitive ovarian cancer cell line. We then explored whether the expression levels in clinical samples relate to letrozole responsiveness for patients with ovarian cancer to assess their value as predictive markers. Materials andMethods Cell culture. The PEO4 and PE014 cell lines were developed at the Edinburgh Cancer Research Center (21). Cell lines were routinely cultured at 37jC, 90% humidity, and 5% CO2 in RPMI 1640 (Life Technologies, Paisley, United Kingdom) containing 10% heat-inactivated FCS, 100 Ag/mL streptomycin, and 100 IU/mL penicillin. Where specified, cells were treated with E2 (Sigma, Poole, United Kingdom), tamoxifen (Sigma), ERa agonist (propyl pyrazole triol, Tocris, Avonmouth, United Kingdom), or ERh-specific agonist (diarylpropionitrile, Tocris) at the concentrations shown. Real-time quantitative PCR. Reverse transcription-PCR was done using a Rotorgene 2000 (Corbett Research, Cambridge, United Kingdom) and a one-step Quantitect reverse transcription-PCR kit (Qiagen, Cowley, United Kingdom) as per protocol. Thermal cycling conditions were 50jC for 30 min and 95jC for 15 min, followed by 45 cycles of 95jC for 15 s, 55jC for 30 s, and 72jC for 45 s. Single-product amplification was confirmed by product melt analyses. PCR primers were designed by Primer 3.0 and Blast searched to check specificity. Specific PCR amplification products were detected by the fluorescent double-stranded DNA-binding dye SYBR Green. Primer sequences used