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Objectives: To elucidate the association between thrombospondin1 (THBS1) expression and TP53 status and THBS1 promoter methylation in epithelial ovarian cancer (EOC). Methods: EOC cell lines with known TP53 status were analyzed for THBS1 gene expression using Affymetrix U133 microarrays and promoter methylation by pyrosequencing. THBS1 mRNA expression was obtained pre- and post-exposure to radiation and hypoxia treatment in A2780 parent wild-type (wt) and mutant (m)TP53 cells. THBS1 expression was compared to tumor growth properties. Results: THBS1 gene expression was higher in cells containing a wtTP53 gene or null TP53 mutation (p = 0.005) and low or absent TP53 protein expression (p = 0.008) compared to those harboring a missense TP53 gene mutation and exhibiting high TP53 protein expression. Following exposure to radiation, there was a 3.4 fold increase in THBS1 mRNA levels in the mTP53 versus wtTP53 A2780 cells. After exposure to hypoxia, THBS1 mRNA levels increased approximately 4-fold in both wtTP53 and mTP53 A2780 cells. Promoter methylation levels were low (median=8.6%; range=3.5-88.8%). There was a non-significant inverse correlation between THBS1 methylation and transcript levels. There was no association between THBS1 expression and population doubling time, invasive capacity, or anchorage-independent growth. Conclusion: THBS1 expression may be regulated via the TP53 pathway, and induced by hypoxic tumor microenvironment conditions. Overall low levels of THBS1 promoter methylation imply that methylation is not the primary driver of THBS1 expression in EOC.

Overexpression of insulin-like growth factor-II (IGF2) is a prominent characteristic of many epithelial ovarian malignancies. IGF2 imprinting and transcription are regulated in part through DNA methylation, which in turn regulates binding of the insulator protein, CTCF, within the IGF2/H19 imprint center. We have shown that IGF2 overexpression in ovarian cancer is associated with hypermethylation of CTCF binding sites within the IGF2/H19 imprint center. The aim of this study was to investigate the methylation and binding capacity of a novel putative CTCF binding motif located intragenic to IGF2 and determine how this relates to IGF2 expression. In 35 primary serous epithelial ovarian cancer specimens, methylation of two CpGs, including one within the core binding motif and another adjacent to this motif, was higher in the 18 cancers with elevated IGF2 expression versus 10 with low expression (avg. 68.2% vs. 38.5%; p92%; avg. 93.2%; N=16). We confirmed binding of CTCF to this region in ovarian cancer cells, as well as the paralog of CTCF, BORIS, which is frequently overexpressed in cancers. The unmethylated CTCF binding motif has insulator activity in cells that express CTCF or BORIS, but not in cells that express both CTCF and BORIS. These intragenic CpG dinucleotides comprise a novel paternal germline imprint mark and are located in a binding motif for the insulator protein CTCF. Methylation of the CpG dinucleotides is positively correlated with IGF2 transcription, supporting that increased methylation represses insulator function. These combined results suggest that methylation and CTCF binding at this region play important roles in regulating the level of IGF2 transcription. Our data have revealed a novel epigenetic regulatory element within the IGF2/H19 imprinted domain that is highly relevant to aberrant IGF2 expression in ovarian malignancies.