Elsevier

Gynecologic Oncology

Volume 104, Issue 2, February 2007, Pages 331-337
Gynecologic Oncology

Emerging roles for PAX8 in ovarian cancer and endosalpingeal development

https://doi.org/10.1016/j.ygyno.2006.08.052Get rights and content

Abstract

Objectives.

Epithelial ovarian carcinomas develop from ovarian surface epithelia that undergo complex differentiation to form distinguishable phenotypes resembling those of the epithelia of the female urogenital regions. While previous studies have implicated regulatory developmental homeobox (HOX) genes in this process, other factors responsible for this differentiation are largely unknown. Aberrant transcriptional expression of PAX8 has been reported in epithelial ovarian cancer, prompting us to initiate the molecular characterization of this master regulatory gene in ovarian cancer development.

Methods.

Immunohistochemistry, immunoblotting and RT-PCR were used to investigate the presence of PAX8 and its protein products in epithelial ovarian cancer subtypes, normal ovarian surface epithelia, ovarian inclusion cysts and normal endosalpingeal epithelia.

Results.

In this report, we confirm microarray results indicating that the transcription factor, PAX8, is highly expressed in epithelial ovarian cancer but absent from the precursor ovarian surface epithelia of healthy individuals. Furthermore, we report that PAX8 is localized to the nucleus of non-ciliated epithelia in simple ovarian epithelial inclusion cysts and in three epithelial ovarian cancer subtypes (serous, endometrioid and clear cell). We also determined that PAX8 is expressed in the non-ciliated, secretory cells of healthy fallopian tube mucosal linings but not in the adjacent ciliated epithelia.

Conclusion.

These findings support the hypothesis that PAX8 plays parallel roles in the development of epithelial ovarian cancer and in the developmental differentiation of coelomic epithelia into endosalpingeal epithelia.

Introduction

Epithelial ovarian carcinoma (EOC) is believed to arise from the single layer of cells found surrounding the ovary, referred to as ovarian surface epithelia (OSE). The OSE are the modified coelomic or peritoneal mesothelia that form a single layer of flat-to-cuboidal cells covering the ovary [1]. Interestingly and unlike most other epithelial derived cancers, the phenotypically uncommitted OSE undergo complex epithelial differentiation as they progress to form polarized epithelia, papillae, cysts and glandular structures, i.e. the cellular differentiation of EOC mimics the development of other differentiated epithelia of the female urogenital and abdominal regions [1].

Extensive microarray data have been generated to establish the gene expression profiles of ovarian carcinoma tissues and cell lines [2], [3], [4], [5], [6], [7], [8], [9], [10], [11], [12], [13], [14], [15], [16], [17], [18], [19]. These studies produced numerous candidate molecular markers for the study and identification of epithelial ovarian cancer. The molecular mechanisms by which the altered expression of these genes contributes to the onset and progression of ovarian cancer are largely unknown.

Marquez et al. recently reported that when compared with normal ovarian epithelial brushings, alterations in microarray gene expression profiles of serous tumors correlated with those in normal fallopian tube (P = 0.0042) but not in other normal tissues [14]. This indicates that EOCs not only imitate the phenotype of other differentiated epithelium of the female reproductive tract, but their gene expression profiles as well. One gene consistently overexpressed in EOC relative to normal ovarian tissue is paired box gene 8 (PAX8, X69699) [2], [10]. Mammalian paired box genes encode a family of 9 transcription factors (PAX1–PAX9) involved in embryogenesis [20]. PAX8 is thought to play a regulatory role in cell fate decisions during the development of the thyroid and kidney [21]. Multiple splice variants of PAX8 have also been identified in Wilms' tumors of the kidney [21]. PAX8 is known to have at least five different splice variants, A–E [22]; however, the significance of these splice variants is presently unknown. By cDNA microarray analysis, PAX8 was shown to be expressed at a significantly higher level in ovarian cancer than in breast cancer [2]. In a subsequent oligonucleotide microarray analysis, PAX8 was reported as one of the top 40 genes that was specifically upregulated > 2-fold in 20 primary serous papillary ovarian carcinomas and 17 ovarian carcinomas metastatic to the omentum when compared to 50 normal ovaries and to over 300 other normal and diseased tissues [10]. However, characterization of PAX8 expression by other experimental methods has been limited [23]. In order to validate the aforementioned microarray results and to further explore the role PAX8 may play in the etiology of EOC, the expression of PAX8 was characterized in four subtypes of EOC tissues, the EOC cell lines BG-1 and OVCAR-3, healthy ovaries and healthy fallopian tubes.

Section snippets

Immunohistochemistry of PAX8 in ovarian tissue

To establish the presence and cellular localization of PAX8 protein in EOC tissue, an immunohistochemical (IHC) screen of archived paraffin embedded samples was performed. Tissues were assayed from 19 serous, 14 endometrioid, 18 mucinous and 2 clear cell subtypes of EOC. Non-neoplastic ovarian tissue from eight patients was assayed as a negative control while thyroid tissue was used as a positive control for PAX8 staining since PAX8 expression is required for proper function of the

Discussion

It is widely accepted that an understanding of developmental evolution is dependant upon knowledge of the “genetic architecture,” i.e. the number of genes and the relative effect of individual genes contributing to a particular cellular or organismal trait [30]. It is our contention that an understanding of the development and evolution of the individual types and subtypes of ovarian cancer is likewise dependent on knowledge of their genetic architecture. While the inappropriate expression of

Human tissues

Archival formalin-fixed, paraffin embedded tissue from ovarian carcinoma of different histological subtypes, non-neoplastic ovarian, fallopian tube and thyroid tissues was obtained from the files of Emory University hospital and Crawford Long hospital, Atlanta, GA. The PAX8 immunohistochemistry screen of these tissues was approved by the Emory University's Institutional Review Board (IRB). Fresh EOC and fallopian tube tissue for protein and RNA isolation was provided by the Ovarian Cancer

Acknowledgments

Research was supported by grants from the Georgia Cancer Coalition to NJB and JFM and a gift in remembrance of Josephine Crawford Robinson for support of the Ovarian Cancer Institute Laboratory. MA was funded by the Georgia Tech Presidential Undergraduate Research Award (PURA) fund. NJB thanks Paul A. Wade and members of his laboratory for invaluable advice and discussions during time spent in his laboratory and Leland W. K. Chung for thoughtful discussions.

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