TITLE : Emerging Roles for PAX 8 in Ovarian Cancer and Endosalpingeal Development

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 flatto-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 [2-19]. 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 over expressed 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. RESULTS 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 two 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 thyroxinproducing follicular epithelia of the mammalian thyroid [24, 25]. Strong nuclear staining of PAX8 in the follicular cells of the thyroid gland was detected, (Figure 1A, B) while the OSE, stroma and granulosa cells lining normal follicles in the ovaries were negative for PAX8 immunoreactivity (Figure 1C, D). Interestingly, the epithelial inclusion cysts of healthy ovaries were positive for PAX8 expression (Figure 1E, F). In one instance of a cyst that had undergone tubal metaplasia, the PAX8 staining was notably intense (Figure 1F, arrowhead). IHC also demonstrated strong positive nuclear expression of PAX8 in the epithelia of ~70% of serous, endometrioid, and clear-cell subtypes of EOC. Representative images of positive PAX8 staining in serous, endometrioid and clear-cell subtypes of EOC are shown in Figure 1G-I. In contrast, mucinous subtypes of EOC display PAX8 expression in only ~10% of screened individuals (Figure 1J). A summary of the results of our IHC of ovarian tissue samples is shown in Table 1. PAX8 expression in fallopian tube secretory epithelium. While screening normal ovarian tissue for the presence of PAX8 by IHC, the presence of fallopian tube (oviduct) sections was detected in several samples. Intense PAX8 staining was detected in the epithelia that line the inner mucosa of the oviduct (Figure 2A). The endosalpingeal lining is composed of simple columnar epithelium consisting of two cell types, ciliated and secretory [26]. The ciliated cells aid in the capture of the ovulated egg for fertilization and the subsequent transport to the uterus. Secretory cells are functionally similar to goblet cells of the intestinal and respiratory tracts, providing lubrication as well as nutrition for the fertilized egg. Upon further examination, it was determined that only the secretory subset of epithelia in the oviduct mucosa was positive for PAX8 expression. Higher magnification exposed complex patterns of ciliated cells separated by PAX8 expressing secretory cells (Figure 2B, C). An endosalpingeal phenotype was also seen in several of the simple inclusion cysts that stained positively for PAX8 in the healthy individuals. More detailed examination of these cysts revealed regions where the ciliated cells were also negative for PAX8 expression (Figure 2D, E). Characterization of the PAX8 gene locus in EOC tissue and cell lines. A translocation identified in a subset of human thyroid follicular carcinomas, t(2;3)(q13;p25), results in the fusion of the DNA binding domains of PAX8 to domains A to F of the peroxisome proliferator-activated receptor g1 (PPARg1) [27]. The resulting fusion protein (~97 kDa) has been hypothesized to play an oncogenic role in thyroid follicular carcinomas. To determine the molecular size of PAX8 present in our samples, immunoblots and RT-PCR were carried out on total protein and ribonucleic acid (RNA) isolated from two phenotypically divergent EOC cell lines (BG-1 [28] and OVCAR-3 [29], described further below), a section of the ampulla from a healthy fallopian tube and the tissue of three individual cases of serous papillary EOC. In the OVCAR-3 cell line, the fallopian tube and the three cases (CA183, -369, -413) of serous papillary ovarian carcinoma examined, the expected 48 kDa protein of the full-length PAX8A isoform was observed by immunoblot analysis (Figure 3A). Furthermore, RT-PCR of these same samples indicated the presence of multiple splice variants of PAX8 in these tissues (Figure 3B). Cloning and sequencing of the RT-PCR products from the OVCAR-3 cell line revealed the presence of PAX8 isoforms A, B, C, and D (Figure 3C). The same four splice variants were also detected in cancer sample 183. In the samples with less abundant RT-PCR product, only isoforms A, B and C were observed. These results do not rule out the presence of isoform D at lower abundance in these tissues. In all samples, the PAX8A isoform was most abundant. Primers for RT-PCR were designed to span the three breakpoints in exons 8, 9 and 10 of PAX8 in the PAX8-PPARg1 fusions identified previously [27] (Figure 3C). The results verify that these exons remain intact in EOC and demonstrate the presence of native PAX8 expression in these samples. These results also confirmed that the expression pattern of PAX8 in fallopian tube tissue is identical to that found in EOC. BG-1 and OVCAR-3 are two independently established EOC cancer cell lines with distinct phenotypes in two-dimensional cell culture. BG-1 cells grow in isolation with elongated, mesenchymal features, whereas OVCAR-3 grows with the characteristic “cobblestone” phenotype of typical epithelial cells (Figure 4A, B). In addition, abundant intracellular vacuoles are found in the perinuclear and cytoplasmic reg