The Role of ALCAM, Activated Leukocyte Cell Adhesion Molecule, in the Aggressive Nature of Breast Cancer Cells, a Potential Connection to Bone Metastasis.

Abstract

Abstract Introduction. ALCAM, activated leukocyte cell adhesion molecule, has been previously reported to be connected to the progression of certain solid tumours. In breast cancer, ALCAM has been shown to be expressed at a reduced level in aggressive tumours (King et al 2004) and is a prominent feature for tumours subsequently developed bone metastasis (Davies et al 2008). In the present study, we investigate the molecular impact of ALCAM on the biological behaviours of breast cancer cells with a particular reference to condition that is linked to bone biological environment.Methods. A mammalian ALCAM expression construct was prepared from normal mammary cDNA bank using a pEF6.V5/His vector. Anti-ALCAM transgenes were prepared based on human ALCAM mRNA structure and cloned into a mammalian expression vector. Suitable cells were transfected with either the expression construct or anti-ALCAM transgene, to create sublines that had differential expression of ALCAM. The growth, migration and invasion of the aforementioned cells, together with their parent and control cell lines were evaluated using a panel of in vitro cell models, in the presence or absence of matrix proteins prepared from human bones. Statistical analysis was Student t test or Mann-Whitney test, where appropriate.Results. Of the 12 human breast cancer lines tested, ZR-75-1 was found to be strongly positive for ALCAM expression, whereas MDA-MB-231 was negative. ZR-75-1 was therefore transfected with the anti-ALCAM transgene. After selection, a subline, ZR-751ΔALCAM was created in which ALCAM expression was knocked out. Likewise, MDA-MB-231 was transfected with ALCAM expression construct, followed by creation of new subline, MDA-MB-231ALCAMexp, a sublined that over-expressed ALCAM. MDA-MB-231ALCAMexp cells showed a marginally slower rate of growth compared with control cells. However, in the presence of bone matrix proteins, MDA-MB-231ALCAMexp showed a significantly reduced rate of growth (growth index 0.62±0.11), p<0.01 vs control cells (1.06±0.15). In contrast, ZR-751ΔALCAM cells showed a faster pace of growth (1.04±0.14) compared with control cells (0.81±0.09). In an extracellular matrix based in vitro invasion assay, it was observed that MDA-MB-231ALCAMexp displayed a significantly reduced invasiveness compared with control cells (p=0.012). ZR-751ΔALCAM cells showed a significantly increased invasiveness vs its control cells (p=0.02). In an electric cell sensing based cell migration assay, we found that MDA-MB-231ALCAMexp cells showed marked reduction in migration compared with its control and parent cells. We have shown that inclusion of bone matrix proteins further reduced the migration speed of MDA-MB-231ALCAMexp cells.Conclusion. The present study provides cellular insight into the role of ALCAM in its involvement in bone metastasis of breast cancer. It is concluded that loss of ALCAM in breast cancer cells facilitates the invasive behaviour of breast cancer and highly levels of ALCAM in the cells have a suppressive role in the aggressive nature of breast cancer cells.1. Davies SR, et al, Oncol Rep., 2008, 19: 555-612. King JA et al, Breast Cancer Res., 2004, 6: R478-87 Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 6174.