Overexpression of L1CAM accompanies acquired endocrine resistance and is associated with the development of an aggressive cell phenotype.

Abstract

Abstract Abstract #3028 Background: Acquired resistance to endocrine therapies is associated with a gain in aggressive features in vitro which may impact on tumour spread in vivo. To identify potential candidate molecules which may promote this adverse cell behaviour, we performed microarray analysis on MCF-7 cell models of fulvestrant resistance ('FasR' cells) applying a commercial algorithm for signalling network discovery. We have subsequently investigated the significance of overexpression of L1CAM, a cell-surface adhesion molecule implicated in a MAPK signalling network and recently associated with tumour metastasis, in FasR cells and in clinical tissue.
 Materials and Methods: A network analysis algorithm was applied to t-test significant Affymetrix gene lists derived from FasR vs. MCF-7 cells (Almac Diagnostics). Subsequent to revealing a prominent L1CAM network, L1CAM gene and protein expression were assessed using RT-PCR, Western blotting and immunofluorescence. Inhibition of L1CAM was achieved using siRNA transfection. Cell invasion and migration were measured by seeding onto Matrigel- or fibronectin-coated microporous membranes respectively. After 48 hours, invasive/migratory cells were fixed, stained and counted. Analysis of cell signalling molecules was determined using Western blotting with phospho-specific antibodies. Clinical significance of L1CAM expression was determined by analysis of L1CAM expression in tumour (n=101) vs. normal (n=23) breast tissue using Q-PCR.
 Results: Development of fulvestrant resistance in MCF7 cells was associated with ER loss and a gain in migratory and invasive capacity in vitro. L1CAM was overexpressed at both gene (p<0.05) and protein (p<0.01) level in FasR cells whereas little or no L1CAM was detectable in MCF7 cells confirming microarray findings. Network and ontological analysis predicted L1CAM lies upstream of an ERK2 network (26 genes; p<0.0001) impinging on transcription factors/regulators which may promote aggressive cell behaviour. In agreement, siRNA-mediated inhibition of L1CAM suppressed the activity of nuclear MAP kinase and reduced the intrinsic migratory and invasive nature of FasR cells in vitro. Preliminary analysis indicated L1CAM expression was inversely associated with ER status (p<0.01) and reduced overall survival (p=0.01).
 Conclusions: These data suggest that L1CAM overexpression associates with emergence of ER-negative fulvestrant resistance in breast cancer, where it may confer a highly motile and invasive phenotype. Additionally, our clinical studies suggest L1CAM may also contribute to inherently aggressive, ER-negative breast cancers. L1CAM signalling may thus provide a new therapeutic target to subvert such aggressive states in vivo. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 3028.