Elsevier

Gynecologic Oncology

Volume 106, Issue 3, September 2007, Pages 461-468
Gynecologic Oncology

Estrogen-regulated gene expression predicts response to endocrine therapy in patients with ovarian cancer

https://doi.org/10.1016/j.ygyno.2007.05.009Get rights and content

Abstract

Objective

To explore the predictive value of estrogen-regulated gene changes as indicators of sensitivity in ovarian cancer patients treated with the aromatase inhibitor Letrozole.

Methods

Expression of a range of proteins was assessed by semi-quantitative immunohistochemistry in tissue sections from the tumors of patients treated with Letrozole. Expression was correlated with clinical response to Letrozole. Corresponding mRNA in ovarian cancer cell lines treated with 17β-estradiol (E2) was measured by quantitative RT-PCR.

Results

In an estrogen receptor (ER)-positive ovarian cancer cell line, quantitative RT-PCR analysis demonstrated that PLAU, VIM, BIGH3, CDH6, FN1, CASP4, KRT4, KRT7, KRT13, TRAM and NGAL were down-regulated and TFF1, TFF3, TRAP1, TFAP4, MYC, CTSD, IL17BR, TOP2A, CCNB1, CCNB2, PDZK1 and UBE2C were up-regulated by E2. The E2 modulation of these genes was reversed by the anti-estrogen tamoxifen and was ERα-dependent.

For ovarian cancer patients treated with Letrozole, we tested the predictive value of the majority of these genes in paraffin sections from their primary tumors by semi-quantitative immunohistochemistry. Significant differences in expression levels of TFF1, TFF3, BIGH3, TRAP1, VIM, TOP2A, PLAU and UBE2C were observed between tumors from CA125 responsive/stable patients as opposed to tumors from patients whose disease progressed, using serum levels of CA125 as an indicator of response. Aromatase expression in the ovarian cancers also differed between these 2 groups of patients.

Conclusion

These results suggest that expression levels of certain proteins in ovarian cancers are estrogen-regulated and could help identify patients who would benefit from endocrine therapy.

Introduction

Epithelial ovarian cancer is the leading cause of death from gynecological malignancy in the western world and the fourth leading cause of cancer death in women worldwide [1]. This is due to the fact that the disease is relatively asymptomatic in its early disease stages with approximately 70% of patients presenting with advanced disease at diagnosis. Primary treatment of ovarian cancer remains surgical debulking of tumor followed by combination chemotherapy. The response rates of relapsed patients especially those with platinum resistance may be low and its associated toxicity impacts on the remainder of the patient's quality of life [2].

Endocrine therapies have proven effective in other hormonally sensitive cancers with relatively little toxicity. Many ovarian cancers express high levels of estrogen receptor alpha (ERα) and experimental studies using preclinical models have demonstrated that ovarian cancer cells that express high levels of ERα are growth responsive to both estrogens and anti-estrogens [3], [4], [5], [6]. This raises the possibility of a therapeutic potential in targeting the ERα in this disease. The anti-estrogen tamoxifen has been used to treat chemo-resistant ovarian cancer [7], [8] and recently, we and others have shown that the aromatase inhibitor Letrozole, which by inhibiting estrogen synthesis reduces the levels of estrogen available to ERα, confers clinical benefit in a sub-group of ovarian cancer patients [9], [10].

In a phase II trial we had recruited ovarian cancer patients (previously treated with chemotherapy) to receive 2.5 mg/day of Letrozole at the time of CA125 relapse of their disease [9]. CA125 responses were evaluable in 54 of these patients with a response being demonstrated in 5 patients and stabilization of CA125 (< 50% increase) in a further 14 patients. Therefore a total of 32% of patients receiving Letrozole achieved clinical benefit. Formalin fixed paraffin-embedded tissues from primary ovarian cancers from these patients were analyzed in the current study.

To further characterize this group of estrogen responsive patients we evaluated the expression of estrogen-regulated genes to see if they might act as predictive markers of response to Letrozole. Candidate genes were identified from a microarray analysis of ER-positive ovarian cancer cells treated with 17β-estradiol (E2) [6] and from studies previously published of genes identified as being modulated by Letrozole in breast cancer clinical trials [11], [12].

Having confirmed the estrogen regulation of candidate genes in an ER-positive, estrogen-sensitive ovarian cancer cell line by RT-PCR, immunohistochemical analysis was undertaken for a subset of these genes to determine whether gene expression levels in histological samples obtained at the time of primary debulking surgery are related to subsequent clinical response to Letrozole.

Section snippets

Cell culture

The PEO4 cell line was developed at the Edinburgh Cancer Research Centre [13]. Cells were routinely cultured at 37 °C, 90% humidity and 5% CO2 in RPMI 1640 (Life Technologies, Paisley, UK) containing 10% heat-inactivated FCS, 100 μg/ml streptomycin, and 100 iu/ml penicillin. Where specified, cells were treated with E2 (Sigma), tamoxifen (Sigma), ERα agonist (propylpyrazole triol (PPT), Tocris, Avonmouth, UK) [14] or ERβ-specific agonist (diarylpropionitrile (DPN), Tocris, Avonmouth, UK) [15] at

Modulation of selected genes by estrogen in estrogen-responsive ovarian and breast cancer cell lines in vitro

To investigate which candidate genes were under estrogen regulation and potentially associated with growth changes, we first investigated a series of genes identified from two sources—microarray analysis and literature selection of breast cancer studies. We have previously reported a microarray analysis of estrogen-regulated genes in an ovarian cancer model [6], and in the present study we have selected those genes showing the most marked changes after estrogen exposure and investigated them

Discussion

In the present study we have identified a series of genes predictive of response to Letrozole in ovarian cancer patients. These had first been validated in an ER-positive estrogen growth responsive ovarian cancer cell line model. The initial group of genes studied had been identified in a microarray analysis and the estrogen sensitivity of these changes was confirmed here by use of QRT-PCR. While TFF1, TRAP1, TFAP4, MYC, CTSD and IL17BR were significantly up-regulated by E2, PLAU, VIM, BIGH3,

Acknowledgment

This work was supported by Cancer Research UK.

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