YY1 imprint status and role in ovarian carcinogenesis

1572 Purpose. YIN-YANG 1 (YY1) encodes a zinc finger protein that exhibits diverse effects on cell function through its ability to both positively and negatively influence transcription of many other genes. YY1 is located ∼0.5 Mb centromeric to the imprinted cluster on human 14q32.2 but its imprint status has not been determined in humans. Our previous microarray analysis of advanced stage serous epithelial ovarian cancers indicated that YY1 is one of the most differentially expressed genes between short term (less then 3y) and long term (more than 7y) survivors, with elevated YY1 expression in the long term survivors (p = 0.00003; Clin. Cancer Res. 2005, 11:3686-3696). The purpose of this study was to determine whether YY1 is subject to genomic imprinting, whether it is deregulated by hypermethylation in epithelial ovarian cancers, and the potential role of YY1 in ovarian cancer cell growth. Procedures. Allelic expression of YY1 was determined by nucleotide sequencing of cDNA from heterozygous individuals. The methylation status of the YY1 promoter was analyzed using MS-PCR and bisulfite sequencing. Transient transfection of siRNA oligos was performed in quadruplicate to suppress YY1 expression in ovarian cell lines and was verified using quantitative real time RT-PCR. The effect of YY1 knockdown was measured using assays for cell proliferation and anchorage-independent growth. Results. Of 12 heterozygous tumor specimens, 11 exhibited biallelic expression of YY1 cDNA. YY1 was also expressed from both parental alleles in a panel of eight human fetal tissues. The YY1 promoter CpG island was unmethylated in 24 normal lymphocyte samples and 87 ovarian tumors, including 24 short term and 21 long term survivors. siRNA-mediated suppression of YY1 resulted in a 13% and 29% reduction in cell proliferation in the ovarian cancer cell lines, HEY (p = 0.02) and HEYA8 (p = 0.006), respectively, as compared to controls which received a non-silencing siRNA oligo. We also observed a substantial inhibition in anchorage independent growth, with HEY and HEYA8 cell lines exhibiting a 72% and 88% reduction in colony number, respectively. Conclusions. Our results indicate that YY1 is not imprinted in humans. Monoallelic YY1 expression observed in one cancer specimen may reflect loss of heterozygosity, since this region of 14q is a known target of LOH in ovarian cancers. Decreased YY1 expression in short term survivors is not due to promoter hypermethylation. The outcome of siRNA-mediated knockdown of YY1 in ovarian cancer cell lines suggests that YY1 is contributing to their oncogenic phenotype. Together, our results show that YY1 has an important role in ovarian carcinogenesis but further work will be required to delineate its apparently divergent functions in vitro and in vivo .