Muc16 carboxyl portion expression alternates PI3K/Akt and EGFR/HER2/ERK signal pathways in human ovarian cells

3435 MUC16 (CA125) is a heavily O-glycosylatedtransmembrane mucin family protein and a cell surface marker for ovarian carcinomas and other gynecologic malignancies, distinguished by highly repetitive sequences in the extracellular domain. MUC16 transfection alters tumor growth pattern in xenograft models and in vitro invasion behavior of MUC16-negative ovarian cancer cells. MUC16 expression is also increased cisplatin resistance but the mechanisms and signaling pathwaysof the MUC16 are unclear. We examine the effect of MUC16 on common signal transduction pathways, such as PI3K/Akt and EGFR/HER2/MAPK pathways, which are associated with malignant transformation, invasion and metastasis. We subcloned the truncated MUC16 carboxyl 114 amino acid and 344 amino acid sequences into phrGFP II- C vector and then transfected the MUC16 expression constructs into MUC16-negative ovarian carcinoma cell lines, SKOV3 and A2780, and a transformed human ovarian epithelial cell line, T80. Stable transfectants were selected and enriched by fluorescence activated cell sorting. Western blotting and immunoprecipitation were performed by using cell lysates of cultured transfectants with antibodies against PI3K, Akt, phosphorylated-Akt Ser473, EGFR, HER2, P-HER2/EGFR Tyr1248/Tyr1173, ERK1/2, P-ERK1/2 Thr202/Tyr204, p38 MAPK and P-p38 MAPK Tyr180/Tyr182, GSK3 beta and P-GSK3 beta Ser9, Bad , P-Bad Ser136 and Ser112. Expression of carboxyl MUC16 stimulated phosphrylation of ERK1/2 compared with phrGFP II-C vector transfectants through upregulating phosphorylated HER2/EGFR Tyr1248/Tyr1173, while this effect was inhibited by EGFR tyrosin kinase inhibitor (TKI), gefitinib or MEK inhibitor, U0126. Carboxyl MUC16 also upregulated Akt phosphorylation as well as its downstream GSK3 beta and Bad phosphorylation, and these effects were inhibited by PI3 Kinase inhibitor, LY294002 , but not gefitinib. These results suggest that the expression of carboxyl MUC16 activates MAPK and PI3K/Akt signaling pathways in SKOV3, A2780 and T80 cells in different mechanisms. The phosphorylation inhibition of ERK by gefitinib but not by LY294002 implies that MUC16 induces MAPK signal upregulation through EGFR/HER2/MAPK in an independent manner from the PI3K/Akt pathway. The biological consequences of the PI3K/Akt and EGFR/HER2/MAPK based signal activation by truncated carboxyl MUC16 are under investigation.