The association between Annexin A2 and epithelial cell adhesion molecule in breast cancer cells.

BACKGROUND: The epithelial cell adhesion molecule (EpCAM) is a type I transmembrane and glycosylated protein, which is overexpressed in many neoplasms. However, EpCAM has no known ligand partners and the mechanisms by which it functions are not fully understood. AIM: This study was performed to discover novel partners of EpCAM, which may provide a better understanding of its functions. METHODS: The membrane fraction of the ERα+ noninvasive breast cancer cell line ZR-75-1 and MCF-7 was extracted and followed by co-immunoprecipitation of EpCAM using C-10, a mouse monoclonal antibody raised against amino acids 24-93 of the EpCAM molecule. As a negative control, MDA-MB-231 and Hs578T were used since they express a negligible amount of EpCAM and are known as EpCAM-/low ERα-/low invasive and tumorigenic breast cancer cell lines. RESULTS: Annexin A2 (ANXA2) was found to be selectively and differentially co-immunoprecipitated with EpCAM in the ERα+ breast cancer cells MCF-7 and ZR-75-1. ANXA2 is a multifunctional protein and known to act as a co-receptor for tissue plasminogen activator (tPA) on the surface of endothelial and cancer cells, thereby affecting fibrinolytic activity and neoangiogenesis as well as invasive and metastatic properties. In this study, the association between EpCAM and ANXA2 was found to affect the activity of tPA. CONCLUSION: This study concludes that ANXA2 co-localizes with EpCAM at the plasma membrane, and the co-localization may have functional implications. Data suggest that EpCAM supports ANXA2 to function as a co-receptor for the tPA, and that EpCAM has a regulatory function on the expression and subcellular localization of ANXA2.

EpCAM associates with endoplasmic reticulum aminopeptidase 2 (ERAP2) in breast cancer cells.

Epithelial cell adhesion molecule (EpCAM) is an epithelial and cancer cell "marker" and there is a cumulative and growing evidence of its signaling role. Its importance has been recognized as part of the breast cancer stem cell phenotype, the tumorigenic breast cancer stem cell is EpCAM(+). In spite of its complex functions in normal cell development and cancer, relatively little is known about EpCAM-interacting proteins. We used breast cancer cell lines and performed EpCAM co-immunoprecipitation followed by mass spectrometry in search for novel potentially interacting proteins. The endoplasmic reticulum aminopeptidase 2 (ERAP2) was found to co-precipitate with EpCAM and to co-localize in the cytoplasm/ER and the plasma membrane. ERAP2 is a proteolytic enzyme set in the endoplasmic reticulum (ER) where it plays a central role in the trimming of peptides for presentation by MHC class I molecules. Expression of EpCAM and ERAP2 in vitro in the presence of dog pancreas rough microsomes (ER vesicles) confirmed N-linked glycosylation, processing in ER and the size of EpCAM. The association between ERAP2 and EpCAM is a unique and novel finding that provides new ideas on EpCAM processing and on how antigen presentation may be regulated in cancer.

Uncoupling of the ERα regulated morphological phenotype from the cancer stem cell phenotype in human breast cancer cell lines.

The CD24(low/-)CD44(+)EpCAM(+) phenotype is associated with breast cancer initiating cells. To investigate if these putative breast cancer stem cell markers are regulated by estrogen receptor alpha (ERα) we have determined the expression levels of EpCAM, CD44 and CD24 in several well characterized breast cancer cell lines. The expression levels of the three adhesion proteins were quantitatively different in the cell lines but the composite CD24(low/-)CD44(+)EpCAM(+) breast cancer stem cell phenotype was shown to exist as a small fraction, between 0.1% and 1.2%, in all breast cancer cell lines tested. Experimental silencing of ERα resulted in a reduced epithelial appearance and partial reduction of CD24 mRNA, while levels of CD44 and EpCAM were unaltered. Moreover, knockdown of ERα led to a change in the morphology of the cells similar to the epithelial to mesenchymal transition phenotype and was associated with decreased E-cadherin expression. Our findings offer new insights into the regulation of the breast cancer stem cell phenotype by ERα and suggest that treatments targeting the breast cancer stem cell adhesion molecules and the ERα pathway may be complementary.