Studies of stem cell and adhesion proteins in breast cancer

Sammanfattning: Breast cancer is the commonest cancer and second leading cause of cancer death in women. It has been shown that breast cancer tumorigenic/stem cell like cells are CD24-/low CD44+EpCAM+. These cells constitute less than 5% of the cells within a cancer and are probably responsible for recurrence and metastasis. In the first paper of the thesis we show that there is uncoupling of the ER? regulated morphological phenotype from the cancer stem cell phenotype in human breast cancer cells. Experimental silencing of ER? resulted in a reduced epithelial appearance and partial reduction of CD24 mRNA, while levels of CD44 and EpCAM were unaltered. Moreover, knockdown of ER? 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 level. Our findings offer new insights into the regulation of the breast cancer cell phenotype by ER?. In the second and third papers we chose to immunoprecipitate the stem cell and cell adhesion protein EpCAM to identify new EpCAM interacting proteins. We have found a candidate EpCAM associated protein named Endoplasmic Reticulum Aminopeptidase 2 (ERAP2). ERAP2 was co-precipitated and colocalized with EpCAM in breast cancer cells both in the cytoplasm/ER and the plasma membrane. We expressed the two proteins in vitro in presence of dog pancreas rough microsomes (ER vesicles) and confirmed N-linked glycosylation of both proteins and the size of EpCAM. We conclude that the association between ERAP2 and EpCAM is a unique and novel finding, providing new ideas on how antigen presentation may be regulated. In the third paper we continue to search for EpCAM associated proteins using coimmunoprecipitation (IP) and mass spectrometry. We found that Annexin A2 co-precipitated with EpCAM. IP, Western blotting and reverse co-IP confirmed the finding. Furthermore both EpCAM and Annexin A2 colocalized in the cytoplasm and cell membrane in EpCAM+ cells. This association requires more studies to show the role of Annexin A2 in breast cancer. In the fourth paper we have assembled a list of genes potentially associated with the breast cancer stem cells and genes that are involved in epithelial-mesenchymal transition (EMT). We performed a gene expression clustering analysis of breast cancer cell lines using cancer cell lines encyclopedia and GenePattern. We found three clusters, one epithelial (cluster alpha), one mesenchymal (beta) and a third (gamma). Both cluster beta and gamma were characterized by relatively low levels of ESR1 (ER?) as compared to cluster alpha. Clustering analysis performed on clinical samples also generated two distinct groups with low ESR1 levels. Further analysis of these three clusters will show whether there are unique gene expression patterns or overlap between them, especially between cluster beta and gamma. Subsequently we have used the same gene list and analyzed different breast cancer datasets present in the Oncomine ® platform to study relationship between EMT and stem cell phenotypes expressing these genes and their correlation with molecular subtypes, and clinical outcome.