Fig 5. Effect of N-glycosylation on MUC16 transformation.

A) Matrigel invasion assay for 3T3 transfected cell lines, phrGFP control vector or MUC16^c114 or MUC16^3(N—A)c114 and MUC16^c114 treated with 0.1 μg/mL Tunicamycin. As seen earlier, MUC16^c114 cell lines were significantly different (p<0.0001) than the phrGFP vector control. The MUC16^3(N—A)c114 cell line was still significantly more invasive (** p = 0.007) compared to the phrGFP vector control. Treatment with the N-glycosylation inhibitor Tunicamycin significantly inhibited matrigel invasion compared to the untreated MUC16^c114 (##p = 0.0003), and MUC16^3(N—A)c114 is highly significant (### = p<0.0001) compared to MUC16^c114, suggesting that N-glycosylation is critical for MUC16-induced matrigel invasion. B) Matrigel invasion assay for 3T3 transfected cell lines compared to the phrGFP control vector. 3T3 cells transfected with MUC16^c114 were treated with media alone, with 5 μg/mL of control pFUSE hIgG1-Fc2 fusion protein, with 5 μg/mL of MUC16^c57-114-pFUSE hIgG1-Fc2 fusion protein, or with 5 μg/mL of ^117-244LGALS3-pFUSE hIgG1-Fc2 fusion protein, as detailed in S4 Fig As seen earlier, the MUC16^c114 cell line was much more invasive than the phrGFP vector control 3T3 cells (p<0.0001), and this increase in invasion was unaffected by exposure to pFUSE vector only protein. In contrast, MUC16^c114 cell line treated with MUC16^c57-114-pFUSE hIgG1-Fc2 fusion protein or ^117-244LGALS3-pFUSE hIgG1-Fc2 fusion protein demonstrated significant (# p = 0.0001) inhibition of matrigel invasion compared to MUC16^c114 control cells. C) Effect of MUC16 expression on ERK/AKT signaling. Phosphorylation of ERK1/2 (pT202/Y204) and AKT (S473) was increased in the 3T3 transfected with MUC16^c114; however, the effect was much diminished in 3T3 cells transfected with the MUC16^3(N—A)c114 vector. Despite the three asparagine—> alanine mutations, Western blot with the anti-MUC16 antibody, 4H11 mAb, showed a higher signal than either the phrGFP vector control or the native MUC16^c114 transfected cells, indicating that the high levels of MUC16^3(N—A)c114 protein is expressed in the transfected 3T3 cells, and surface expression was confirmed in S2 Fig β-Actin normalized densitometry quantification values are shown below each Western blot in the figure. D) MUC16-positive tumor growth in athymic nude mice. Two million tumor cells were introduced into the flank of 20 nu/nu mice, and the mice were observed for tumor formation. Tumors were measured by calipers twice weekly. The differences in mean tumor volume were significantly greater for mice bearing MUC16^c114 tumors (p<0.0001). As seen earlier, 3T3 MUC16^c114 transfectant was highly significant at *** p<0.0001 compared to the phrGFP control vector. However, MUC16^3(N—A)c1143T3 transfectants did not show any significance over phrGFP vector control 3T3 cells, indicating that the mutations of N-glycosylation dramatically decreased in vivo tumor growth and invasion.