Abstract B52: Annexin A6 promotes cellular attachment and invasiveness of breast cancer cells
Authors: Sakwe Amos M. , Adhiambo Christine , Ochieng Josiah 
Published: May 2012

Abstract
Annexin A6 (ANXA6) is a member of a family of Ca2+-dependent phospholipid-binding proteins capable of associating with negatively charged membranes in its Ca2+-bound state. Although the functions of this protein are yet to be clearly defined, various physiological roles have been proposed, including a role in the regulation of cell growth and tumorigenesis. We previously showed that ANXA2, ANXA4 and ANXA6 are receptors of the serum factor fetuin-A on the surface of breast carcinoma cells. To determine which of these annexins is the bona fide cell surface receptor for fetuin-A, we have now examined the surface expression of ANXA2 and ANXA6 in the fetuin-A expressing hepatocellular carcinoma cell line HepG2 and in the invasive breast carcinoma cell line BT549. By glycerol gradient centrifugation of the post nuclear supernatants of HepG2 cells briefly cultivated at high extracellular Ca2+, we show that while ANXA2 predominantly associated with heavy membranes corresponding to the endogenous fetuin-A profile, the total membrane-associated ANXA6 on the contrary, diminished. Analysis of the EGTA-solubilized surface-associated proteins from HepG2 and BT549 cells revealed that ANXA6 is predominantly associated with the cell surface suggesting that it is externalized. Further analysis of the secreted proteins by differential velocity centrifugation and in particular the nanovesicle-associated (exosomal) secreted proteins confirmed that while secreted ANXA2 was barely detectable, ANXA6 was abundantly secreted via the exosomal pathway. From these data we hypothesized that the surface associated ANXA6 may be the major fetuin-A receptor on the surface of the invasive BT549 breast cancer cells and that it may be important in the growth, attachment and migration of these tumor cells. Using ANXA6 targeted shRNA we show that down regulation of ANXA6 inhibited the attachment of these cells to collagen Type I, Collagen Type IV and to the serum factor fetuin-A but attachment to and spreading on fibronectin was not affected. Based on the detection of vinculin and focal adhesion kinase, we demonstrate the complete absence of adhesion plaques in the ANXA6-depleted cells compared to the parental BT549 cells. This supports our observation that the ANXA6-depleted BT549 cells lost their ability to spread and hence are predominantly spindle-shaped; Moreover, the ANXA6-depleted BT549 cells proliferate faster than the parental cells; they grow in three-dimensional cultures as spheroid colonies resembling the ANXA6-negative MCF-10A acini rather than as stellate colonies produced by the parental cells. Finally, ANXA6 depletion in BT549 cells inhibited their migration and in vitro invasion of matrigel. Together our data demonstrate that ANXA6 expression in this metastatic breast cancer cells correlates with the invasive phenotype and that its association with the surface of these breast cancer cells modulates at least in part, attachment of these cells to the ECM and their overall morphology.
Citation Information: Cancer Res 2009;69(23 Suppl):B52.