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Spontaneous transformation of murine oviductal epithelial cells: A model system to investigate the onset of fallopian-derived tumors

ABSTRACT: High grade serous ovarian cancer (HGSOC) can originate from fallopian tube epithelium (FTE) and ovarian surface epithelium (OSE). We report the application of unique spontaneous model that mimics cellular aging for understanding the origin and progression of HGSOC from oviductal epithelium. Oviductal epithelium is equivalent to human FTE. Serial passaging of the outbred mouse CD1 oviductal cells (MOE low) to MOE high produced transformed cells that lead to benign tumors. To understand the altered molecular signaling pathways in MOEhigh cells versus MOElow cells, we performed RNA sequencing. Total RNA was extracted from MOELOW (passages 8, 9, & 10) and MOEHIGH (passages 90, 103, & 113) cells. Each total RNA sample had ribosomal RNA removed using TruSeq Stranded Total RNA with Ribo-Zero (Illumina, San Diego, CA). Strand-specific libraries were constructed and quantitated using Qubit, and cDNAs verified by qPCR. qRT–PCR validation was performed using SYBR Green assays. Samples were barcoded and sequenced using Illumina HiSeq2500 sequencing. The reads were aligned to the Mus musculus genome (mm10) using TopHat, version and were used to determine the expression of known mmu10 gene annotations from the University of California-Santa Cruz website using Cuffdiff version. By merging the individual transcript from Cuffdiff into a single gene annotation file, we determined the differential expression analysis. By applying a false discovery rate (FDR)-adjusted p-value, where significance was set to p ≤ 0.05, statistically significant differential expression was determined. Furthermore, pathway analysis was performed on transcript lists from both cell lines using GeneCoDis to identify the KEGG and Panther pathways that are significantly different between MOELOW and MOEHIGH cell lines. We find that the splicesome, RNA transport, the cell cycle, and DNA replication were the most highly upregulated pathway whereas the repressed pathways included processing in the endoplasmic reticulum, focal adhesion, and the lysosome. RNA sequencing revealed that p53 in MOELOW and MOEHIGH cells was not mutated; however, MOEHIGH cells had a significant upregulation of a splice variant of p53. The splice variant behaved like wild-type on few targets and missense on some transcriptional targets by qRT-PCR. This study provides a framework for the application of comprehensive chromatin profiling towards characterization of diverse mammalian cell populations. This model provides a framework to uncover a step-wise progression of tumor formation from an oviductal origin to be compared to human disease.

ORGANISM(S): Mus musculus

PROVIDER: GSE70016 | GEO | 2016/06/01

SECONDARY ACCESSION(S): PRJNA287492

REPOSITORIES: GEO

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Project description:High grade serous ovarian cancer (HGSOC) can originate from fallopian tube epithelium (FTE) and ovarian surface epithelium (OSE). We report the application of unique spontaneous model that mimics cellular aging for understanding the origin and progression of HGSOC from oviductal epithelium. Oviductal epithelium is equivalent to human FTE. Serial passaging of the outbred mouse CD1 oviductal cells (MOE low) to MOE high produced transformed cells that lead to benign tumors. To understand the altered molecular signaling pathways in MOEhigh cells versus MOElow cells, we performed RNA sequencing. Total RNA was extracted from MOELOW (passages 8, 9, & 10) and MOEHIGH (passages 90, 103, & 113) cells. Each total RNA sample had ribosomal RNA removed using TruSeq Stranded Total RNA with Ribo-Zero (Illumina, San Diego, CA). Strand-specific libraries were constructed and quantitated using Qubit, and cDNAs verified by qPCR. qRTâPCR validation was performed using SYBR Green assays. Samples were barcoded and sequenced using Illumina HiSeq2500 sequencing. The reads were aligned to the Mus musculus genome (mm10) using TopHat, version and were used to determine the expression of known mmu10 gene annotations from the University of California-Santa Cruz website using Cuffdiff version. By merging the individual transcript from Cuffdiff into a single gene annotation file, we determined the differential expression analysis. By applying a false discovery rate (FDR)-adjusted p-value, where significance was set to p â¤ 0.05, statistically significant differential expression was determined. Furthermore, pathway analysis was performed on transcript lists from both cell lines using GeneCoDis to identify the KEGG and Panther pathways that are significantly different between MOELOW and MOEHIGH cell lines. We find that the splicesome, RNA transport, the cell cycle, and DNA replication were the most highly upregulated pathway whereas the repressed pathways included processing in the endoplasmic reticulum, focal adhesion, and the lysosome. RNA sequencing revealed that p53 in MOELOW and MOEHIGH cells was not mutated; however, MOEHIGH cells had a significant upregulation of a splice variant of p53. The splice variant behaved like wild-type on few targets and missense on some transcriptional targets by qRT-PCR. This study provides a framework for the application of comprehensive chromatin profiling towards characterization of diverse mammalian cell populations. This model provides a framework to uncover a step-wise progression of tumor formation from an oviductal origin to be compared to human disease. Examination of altered molecular signaling pathways in 2 cell types.

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.

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Spontaneous transformation of murine oviductal epithelial cells: A model system to investigate the onset of fallopian-derived tumors

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