Project description:Ovarian cancer is the fifth leading cause of cancer death among US women. Evidence supports the hypothesis that high-grade serous ovarian cancers (HGSC) may originate in the distal end of the fallopian tube. Although a heterogeneous disease, 96% of HGSC contain mutations in p53. In addition, the “p53 signature”, or overexpression of p53 protein (usually associated with mutation), is a potential precursor lesion of fallopian tube derived HGSC suggesting an essential role for p53 mutation in early serous tumorigenesis. To further clarify p53-mutation dependent effects on cells, murine oviductal epithelial cells (MOE) were stably transfected with a construct encoding for the R273H DNA binding domain mutation in p53, the most common mutation in HGSC. Mutation in p53 was not sufficient to transform MOE cells, but did significantly increase cell migration. A similar p53 mutation in murine ovarian surface epithelium (MOSE), another potential progenitor cell for serous cancer, was not sufficient to transform the cells nor change migration suggesting tissue specific effects of p53 mutation. Microarray data confirmed expression changes in pro-migratory genes in p53R273H MOE compared to parental cells, which could be reversed by suppressing Slug expression. Combining p53R273H with KRASG12V activation caused transformation of MOE into high-grade sarcomatoid carcinoma when xenografted into nude mice. Elucidating the specific role of p53R273H in the fallopian tube will improve understanding of changes at the earliest stage of transformation and could help develop chemopreventative strategies to prevent the accumulation of additional mutations and reverse progression of the “p53 signature” thereby, improving survival rates. We used a microarray to determine global changes in gene expression as a result of the p53 mutation specifically in mouse oviductal epithelium. Murine oviductal cells (MOE) were obtained from Dr. Barbara Vanderhyden at the University of Ottawa. Stable cell lines were generated using antibiotic resistant plasmids containing p53 R273H (Addgene, plasmid: 16439, donated by Dr. Vogelstein, Johns Hopkins University school of Medicine, Baltimore, MD) or pCMV-Neo (Origene PCMV6XL4, Rockville, MD). Total RNA was extracted from cell pellets collected from consecutive passages.

Project description:Recent evidence suggests that ovarian high-grade serous carcinoma (HGSC) originates from the epithelium of the fallopian tube. However, most mouse models are based on the previous prevailing view that ovarian cancer develops from the transformation of the ovarian surface epithelium. Here, we report the extensive histological and molecular characterization of the mogp-TAg transgenic mouse, which expresses the SV40 large T-antigen (TAg) under the control of the mouse müllerian-specific Ovgp-1 promoter. Histologic analysis of the fallopian tubes of mogp-TAg mice identified a variety of neoplastic lesions analogous to those described as precursors to ovarian HGSC. We identified areas of normal appearing p53-positive epithelium that are similar to “p53 signatures” in the human fallopian tube. More advanced proliferative lesions with nuclear atypia and epithelial stratification were also identified that were morphologically and immunohistochemically reminiscent of human serous tubal intraepithelial carcinoma (STIC), a potential precursor of ovarian HGSC. Beside these noninvasive precursor lesions, we also identified invasive adenocarcinoma in the ovary of 56% of the mice. Microarray analysis revealed several genes differentially expressed between the fallopian tube of mogp-TAg and WT C57BL/6. One of these genes, Top2a, which encodes topoisomerase II-alpha, was shown by immunohistochemistry to be concurrently expressed with elevated p53 and specifically elevated in mouse STICs, but not in surrounding tissues. TOP2A protein was also found elevated in human STICs, low-grade, and high-grade serous carcinoma. The mouse model reported here displays a progression from normal tubal epithelium to invasive HGSC in the ovary, and therefore closely simulates the current emerging model of human ovarian HGSC pathogenesis. This mouse therefore has the potential to be a very useful new model for elucidating the mechanisms of serous ovarian tumorigenesis, as well as for developing novel approaches for the prevention, diagnosis, and therapy of this disease. Keywords: transgenic mouse model, ovarian cancer, fallopian tube, intraepithelial carcinoma 6 mouse fallopian tubes (FT) were analyzed with experimental repeats; 3 wildtype C57BL6 mice (FT) and 3 transgenic mogp-TAg (FT), with one set of each at 7, 8 and 9 weeks of age.

Project description:The fallopian tube epithelium is one of the potential sources of high-grade serous ovarian cancer (HGSC). The use of estrogen only hormone replacement therapy increases ovarian cancer risk. Despite estrogenâ??s influence in OVCA, selective estrogen receptor modulators (SERMs) typically demonstrate only a 20% response rate. This low response could be due to a variety of factors including the loss of estrogen receptor signaling or the role of estrogen receptor signaling in different potential cell types of origin. The response of fallopian tube epithelium to SERMs is not known, and would be useful when determining therapeutic options for tumors that arise from this cell type, such as high-grade serous cancer. Using normal murine derived oviductal epithelial cells (mouse equivalent to the fallopian tube) estrogen receptor expression was confirmed and interaction with its ligand, estradiol, triggered mRNA and protein induction of progesterone receptor (PR). The SERMs 4-hydroxytamoxifen, raloxifene and desmethylarzoxifene, functioned as estrogen receptor antagonists in the oviductal cells. Cellular proliferation and migration assays suggested that estradiol does not significantly impact cellular migration and increased proliferation in CD1, but not in FVB derived cell lines. Further, using RNAseq, the oviduct specific transcriptional genes targets of estrogen and 4-hydroxytamoxifen signaling were determined and validated. The RNA-seq revealed enrichment in proliferation, anti-apoptosis, calcium signaling and steroid signaling processes. Finally, the ER and PR receptor status of a panel of HGSC cell lines was investigated highlighting the need for better models of estrogen responsive HGSC cell lines. Murine oviductal epithelial cells from the FVB background were hormone starved for 48 hours (with a media change after 24 hours), then treated in triplicate with solvent control (DMSO) (0.1%), 1 nM 17-betaestradiol or 100 nM 4-hydroxytamoxifen for 24 hours. Following treatment, RNA was was isolated, libraries were prepped and sequenced using the Illumina HiSeq 2500 platform.

Project description:Recent evidence suggests that ovarian high-grade serous carcinoma (HGSC) originates from the epithelium of the fallopian tube. However, most mouse models are based on the previous prevailing view that ovarian cancer develops from the transformation of the ovarian surface epithelium. Here, we report the extensive histological and molecular characterization of the mogp-TAg transgenic mouse, which expresses the SV40 large T-antigen (TAg) under the control of the mouse müllerian-specific Ovgp-1 promoter. Histologic analysis of the fallopian tubes of mogp-TAg mice identified a variety of neoplastic lesions analogous to those described as precursors to ovarian HGSC. We identified areas of normal appearing p53-positive epithelium that are similar to “p53 signatures” in the human fallopian tube. More advanced proliferative lesions with nuclear atypia and epithelial stratification were also identified that were morphologically and immunohistochemically reminiscent of human serous tubal intraepithelial carcinoma (STIC), a potential precursor of ovarian HGSC. Beside these noninvasive precursor lesions, we also identified invasive adenocarcinoma in the ovary of 56% of the mice. Microarray analysis revealed several genes differentially expressed between the fallopian tube of mogp-TAg and WT C57BL/6. One of these genes, Top2a, which encodes topoisomerase II-alpha, was shown by immunohistochemistry to be concurrently expressed with elevated p53 and specifically elevated in mouse STICs, but not in surrounding tissues. TOP2A protein was also found elevated in human STICs, low-grade, and high-grade serous carcinoma. The mouse model reported here displays a progression from normal tubal epithelium to invasive HGSC in the ovary, and therefore closely simulates the current emerging model of human ovarian HGSC pathogenesis. This mouse therefore has the potential to be a very useful new model for elucidating the mechanisms of serous ovarian tumorigenesis, as well as for developing novel approaches for the prevention, diagnosis, and therapy of this disease. Keywords: transgenic mouse model, ovarian cancer, fallopian tube, intraepithelial carcinoma

Project description:Mutations in BRCA1 and BRCA2 genes confer an increased lifetime risk for breast and ovarian cancer. Ovarian cancer risk can be decreased by risk-reducing salpingo-oophorectomy (RRSO). Studies on RRSO material have altered the paradigm of serous ovarian cancer pathogenesis. The purpose of this study was to identify candidate genes possibly involved in pathogenesis of serous ovarian cancer by carrying out a microarray analysis of differentially expressed genes in BRCA1/2- mutation positive ovarian and fallopian tube epithelium derived from RRSO surgery. Freshly frozen ovarian and fallopian tube samples from nine BRCA1/2 mutation carriers scheduled for RRSO were prospectively collected in comparison with five mutation-negative control patients undergoing salpingo-oophorectomy for benign indications. Microarray analysis of genome-wide gene expression was performed on ovarian and fallopian tube samples from BRCA1/2 and control patients. The validation of microarray data was performed by quantitative real-time polymerase chain reaction (qRT-PCR) in selected cases of RRSO samples, and also high grade serous carcinoma samples collected from patients with BRCA phenotype. From 22,733 genes, 454 transcripts were identified that were differentially expressed in BRCA1/2 mutation carriers when statistically compared to controls pooling all ovarian and fallopian tube samples together. Of these, 299 genes were statistically significantly downregulated and 155 genes were upregulated. Differentially expressed genes in BRCA1/2 samples reported here might be involved in serous ovarian carcinogenesis and provide interesting targets for further studies. Both fallopian tube and ovarian samples were collected from each BRCA1/2 mutation carrier resulting in eighteen mutation positive adnexal samples. Both fallopian tube and ovarian control samples were collected from one control patient while either ovarian or fallopian tube sample was available from four control patients, respectively, resulting in 6 adnexal control samples. High quality RNA was available from nine BRCA1/2-mutation positive ovarian and eight BRCA1/2-mutation positive fallopian tube samples and from three control ovarian and three control fallopian tube samples.

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Mutant p53 in fallopian tube epithelium and high-grade serous cancer formation

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