Abstract 3045: Glycosylation dependence in MUC16/CA125 expression in ovarian cancer..

Abstract

Abstract CA125 antigen is an established characteristic of human ovarian cancer. Yet its function remains largely unknown. The cloning of MUC16, encoding the CA125 antigen, has opened the door to additional functional exploration. MUC16 has a molecular size of over 14,000 amino acids and consists of a 31 amino acid cytoplasmic domain, a 25 amino acid transmembrane region, a 58 amino acid ectodomain (proximal to the putative cleavage site) and an external domain of up to 60 tandem repeats. As previously reported, as little as 114 AA of MUC16 from the carboxyterminus will traffic to the membrane and induce an altered, more aggressive phenotype, and longer elements from the carboxyterminus with multiple CA125 antigenic tandem repeats were not substantially different. When A2780, SKOV3 or 3T3 cell lines expressed these short elements (both the c114 and the c344) of the MUC16 gene carboxyterminus, they had substantial increases in soft agar colony formation, matrigel invasion and increased phosphorylation of AKT and ERK signaling. Associated with these alterations in signal transduction are changes in gene expression including upregulation of fibronectin, IL-1β, MMP7, and MMP9. While expression of these MUC16 fragments does not change in vitro growth, when placed in nu/nu mice, the transformed tumors were more aggressive with a more disseminated phenotype and statistically significant more rapid tumor growth. Examination the c114 fragment for functional areas has been undertaken. Deletion of the ectodomain (c80) completely blocks these effects and is similar to the vector only controls for invasion, soft agar colony formation and xenograft growth. In contrast, loss of the intracellular domain (c86) has no effect and is similar to the c114 transformants. However, coprecipitation experiments did not yield specific partners. We hypothesized that the ectodomain function was related to glycosylation. Cell surface MUC16 interacts with key growth factor receptors via lectins like Galectin 3 to alter cell surface expression, receptor recycling, and phosphorylation to promote growth and invasion. Using our paired isogenic, MUC16+/- cell lines (3T3 and SKOV3), N-glycosylation site mutant were created and these mutants inhibited the matrigel invasion, lowered activation of AKT and ERK and also inhibited tumor growth in nude mice. We explored this phenomena and created shRNA knock down of MGAT5 (the first enzyme in protein glycosylation) or Galectin 3 (amplified in many high grade serous cancers) and expressed in SKOV3 and 3T3 cell lines. These lines completely reversed the effect of MUC16 expression of matrigel invasion, soft agar growth, ERK /AKT activation, EGFr phosphorylation, and xenograft growth, confirming that intact glycosylation is required for MUC16 effects on oncogene activation, matrigel invasion and in vivo growth. Mucins expression in malignant cells are associated with an enhanced tumor aggressiveness through N-glycosylation. Citation Format: Dharmarao Thapi, Xiu Jun Yan, Nestor Rosales, David R. Spriggs. Glycosylation dependence in MUC16/CA125 expression in ovarian cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3045. doi:10.1158/1538-7445.AM2013-3045