P2-01-17: L1-CAM Promotes Adhesion of Breast Cancer Cells to the Endothelium.

Abstract

Abstract Background: Overexpression of the adhesion molecule L1 -CAM (L1) in breast cancer is correlated with nodal involvement, high grading, and a shorter recurrence-free and overall survival, but the mechanism leading to this effect is poorly understood. L1 is also expressed in endothelial cells promoting the interaction between lymphatic cells and endothelia via homophilic L1-L1 interaction. This mechanism might also mediate adhesion of breast cancer cells to endothelia and thus promote metastasis. To examine this likely role of L1, the impact of L1 expression in breast cancer cells on their adherence to human pulmonary microvascular endothelial cells (HPMEC) was investigated and potential L1 ligands on these cells were identified. Methods: MDA-MB231-Fra2 breast cancer cells expressing high levels of L1 were stably transfected with shRNA vectors to generate two clones with strong L1 downregulation (L1low clones). Adherence of these clones to endothelial cells was studied in dynamic cell-flow adhesion assays in channels seeded with HPMEC simulating in vivo-conditions. Rolling or adherent cells per minute were counted using CapImage software. For the cell-flow assay HPMEC were activated with TNFα. In order to identify the L1 ligand, endothelial cells were partly pre-incubated with blocking antibodies directed to the potential binding partners L1, ALCAM, ICAM-1 and E-selectin before interaction with tumor cells. In additional experiments, flow chambers were coated with recombinant L1-CAM, ICAM1, ALCAM or E-selectin, and the adherence of MDA-MB231 cells with high or low L1 expression to these proteins was investigated. Results: Adhesion of MDA-MB231 cells to activated HPMEC was significantly higher in L1high cells compared to L1low clones, where the number of adherent cells was only 40–50% of the L1high control (p=0.025; p=0.035). Blocking experiments showed that the adherence of L1high cells could be reduced by antibodies directed to ALCAM (p=0.0007), but not to ICAM1. Anti-L1 antibodies had a significant effect only in passages of endothelial cells which showed L1 expression, whereas E-selectin blocking strongly diminished adherence of breast cancer cells irrespective of their L1 expression. In addition, L1low clones showed significantly lower adhesion to recombinant L1, ALCAM and E-selectin proteins compared to the parental cells. Conclusion: Our experiments indicate that L1 expression in breast cancer cells leads to an increased adherence to activated endothelial cells via homophilic (L1-L1) or heterophilic (L1-ALCAM) interactions. This mechanism is a possible explanation for the increased metastatic potential and poor prognosis in L1-positive carcinomas observed in vivo. Our results suggest that this adhesion molecule might be a suitable target for therapeutic interventions. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P2-01-17.