Proteomic analysis of a preneoplastic phenotype in ovarian surface epithelial cells derived from prophylactic oophorectomies

Abstract

Objective.

To study the pattern of protein expression associated with a predisposition to develop ovarian cancer.

Methods.

Prophylactic oophorectomy is used to prevent ovarian carcinoma in high-risk populations who have a strong family history of breast/ovarian cancer. In ovarian specimens of these women, the ovarian surface epithelium (OSE), which is tissue of origin of epithelial ovarian cancer, often shows altered morphology, growth patterns and differentiation features that are believed to be preneoplastic. This study has used a proteomic approach, based on two-dimensional gel electrophoresis and mass spectrometry, to compare the protein profiles of OSE from women with a history of familial ovarian cancer (FH-OSE), i.e., at least two first-degree relatives with such cancer and/or testing positive for BRCA1 mutations, to those without such history (NFH-OSE).

Results.

Of >1500 protein spots, there were 8 proteins whose levels were significantly altered in FH-OSE. Three were known ovarian tumor associated proteins, others were novel changes. A number of the alterations seen were accompanied with protein modifications and have not been previously reported. There was a predominance of sequences related to the stress response pathway. Differential expression of selected genes was confirmed by Western blotting and real-time reverse transcription polymerase chain reaction.

Conclusions.

Our findings define the OSE phenotype of women at a high risk of developing ovarian cancer. Protein alterations seen in these tissues may represent an early, irreversible, non-mutational step in ovarian epithelial neoplastic progression and may be potential early and sensitive markers for the evaluation of cancer risk.

Introduction

The epithelial ovarian carcinomas, which originate from the ovarian surface epithelium (OSE), are the prime cause of death from gynecological malignancies in European and North American women. One reason for the high mortality of ovarian cancer is that, unlike many other cancer types, ovarian cancer is notorious for its insidious properties in early stages. Close to 70% of patients present with the disease spread beyond the pelvis, resulting in a long-term survival rate of only 29% [1]. Indeed, if a woman is diagnosed with an early stage (stage I) ovarian cancer, the survival rate is close to 90% without altering current therapeutic approaches. Thus, it is urgently needed to understand the early events and etiology of the disease. Although there are many genetic and environmental factors which can influence a woman's risk of getting breast and ovarian cancer, a strong family history is by far the most important and best-defined epidemiological risk factor.

Recently, cancer-prone women with an inherited predisposition to ovarian cancer, often BRCA1 (a candidate tumor suppressor in breast and ovarian carcinomas) mutation carriers, have undergone prophylactic oophorectomies as a preventive approach [2], [3]. The high prevalence of ovarian cancer in cancer-prone women provides an excellent model to uncover new players in early ovarian carcinogenesis and, perhaps, means of defining and detecting risk factors at an early, curable stage. Importantly, several studies have identified microscopic benign-to-malignant morphologic features in these ovarian specimens, suggesting the existence of preneoplastic phenotypes in the cells [4], [5], [6], [7]. However, little is known about the molecular changes that are associated with or account for the preneoplastic morphologic changes.

Over 90% of ovarian cancers are thought to arise from the ovarian surface epithelium (OSE), which is a simple epithelial layer covering the ovaries. Since OSE is a minute part of the intact ovary, only limited amounts of tissue can be obtained from a single specimen. Thus, the ability to culture OSE provides an opportunity of obtaining large enough quantities of relatively pure populations of ovarian epithelial cells for in vitro studies [8], [9]. Although several previous studies have identified genes differentially expressed in ovarian cancer, our analysis represents a better approximation of the preneoplastic stage of ovarian cancer development. The majority of previous analyses were performed in ovarian cancer cells and compared with normal OSE cells [10], [11], [12], whereas we chose to compare overtly normal OSE cells of high-risk individuals to OSE cells of the general population. We and others have revealed the presence of premalignant histologic and/or biologic alterations in OSE cells from prophylactically removed ovaries of high-risk individuals [13], [14], [15], [16], [17].

This study has used a proteomic approach to compare the protein profiles of OSE. This powerful analytical technology, in contrast to array methodologies as in previous comparative gene studies, is able to provide an unbiased and comprehensive expression profiling without prior knowledge of the expressed proteins in the starting material [10], [11], [12]. It also offers the advantages of detection at the functional level of protein expression and the ability to also detect posttranslational modifications of proteins, which can easily be missed by transcriptional profiling. Differential expression of selected genes observed in this study was further confirmed by Western blotting and real-time reverse transcription polymerase chain reaction.