MUC16 is a membrane-bound mucin that extends from the surface of ovarian cancer cells. Recent studies suggest that MUC16 influences carcinogenesis and metastasis of ovarian cancer cells. It is expected that protein-protein interactions play a crucial role in MUC 16 signaling and oncogenic transformation.

To define the mandatory molecular partners for MUC16 signaling and transformation, we investigated potential protein binding partners, using a classic yeast two-hybrid screening. The 80AA MUC16-CDΔ102 construct (ectodomain deletion mutant) was cloned into the bait plasmid pGBKT7 and transformed into Y2HGold yeast cells, allowing expression of MUC16-CDΔ102 fused to the Gal4 DNA-binding domain (BD). A SKOV-3 cDNA library was generated and cloned into the prey plasmid pGADT7 and transformed into yeast cells, allowing expression of cDNAs fused to the Gal4 activation domain (AD). Bait and prey yeast cells were allowed to bind to potential partners and bringing the BD and AD into proximity to each other, which then activated transcription of three reporter genes. An array of diploid yeast clones was produced, by co-culturing the two strains overnight, each co-expressing the bait with a different library prey protein. The clone pool was plated on selective media (basic SD-/Trp with Agar) and incubated to screen for individual clones that express the appropriate reporter genes and markers, indicating the presence of interacting hybrid protein pairs. Prey plasmids were then isolated from yeast cells and DNA fragments of the positive clones were amplified by PCR to determine the interacting binding partner.

Seven potential binding proteins (no. of colonies), i.e. TTC1 (4), DARS (1), MAPK1IP1L (1), FAM175B (1), SEPT2 (1), CAMLG (4), and SKAP2 (3), were identified. Protein-complex immuno-precipitation analysis is planned to confirm the interactions between the seven potential binding proteins in human ovarian cancer cell lines, in order to verify that each identified member of the protein complex was a valid identification.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1946. doi:10.1158/1538-7445.AM2011-1946