Analysis of the secreted proteomes of human mammary carcinoma cells

Abstract

During lactation, the mammary epithelial cell plays a critical role in the production of the protein content of milk, which provides nutrients and bioactives that influence both the development and protection of the newborn. In relation to the development of breast cancer, the majority of human tumours arise from epithelial tissues. Consequently, the study of proteins secreted from neoplastic mammary epithelial cells is of interest to various fields of biological research, and is an attractive model system for studying the mammary gland. One of the advantages of such a model system is that the proteins identified are specific to the cell line being cultured, and there is no need to eliminate other contaminating cell types that can make it difficult to distinguish the origins of the proteins identified. The proteomic study of secreted proteins, employing a cell culture model system, provides an alternative sample type for the initial mining required for potential biomarker discovery, as well as allowing for the identification of proteins involved in tumour growth, maintenance, and progression. More biologically-complex mixtures such as serum, nipple aspirate fluid and milk, may be less suitable for this purpose because high-abundance proteins may mask less abundant, yet potentially interesting proteins. The project analysed the secreted proteomes of human mammary carcinoma cell lines, using a combination of 2-D electrophoresis (2-DE) and mass spectrometry. Four breast carcinoma cell lines were studied, the estrogen-receptor (ER)-positive cell lines T47D and MCF7, and the ER-negative cell lines MDA-MB-231 and Hs578T. Several pilot studies were also performed using a fifth mammary cell line, Hs578Bst, which is derived from nonneoplastic tissue. However, further studies were not possible with this cell line due to the difficulty in obtaining enough secreted proteins for large-scale proteomic studies. The analysis of secreted protein fractions from the four breast carcinoma cell lines led to the identification of 823 proteins, representing 193 unique proteins. Secreted proteins identified included a number of bioactive and immune-modulating proteins. Other proteins were related to the cancer phenotype of the cells. A large number of proteases and protease inhibitors were identified as secreted from MDA-MB-231 cells, which reflects the highly invasive potential of this cell line. The secreted fractions from Hs578T cells contained a high number of proteins from the extracellular matrix (ECM), with essential roles in cell adhesion, spreading and migration, and may represent the acquisition of mesenchymal characteristics. Additionally, several known components of milk were identified, which did not include the major milk proteins. In several cases, proteins were identified which have not previously been detected in breast carcinoma tissue or secretory fractions, such as acyl- CoA-binding protein, pentraxin-related protein PTX3, cl9orflO/IL-25 and follistatin-related protein 1, and may demonstrate new roles for these proteins in breast cancer. An additional aim was to establish a baseline from which the effects of various bioactive factors that may influence the secreted proteome could be investigated. Preliminary studies investigated the effects of retinoic acid (RA) and transforming growth factor-p (TGF-P) on the secreted proteome pattern of two of cell lines. Based on these studies, it was concluded that 2-DE analysis of secreted proteins is not an ideal platform for bioassay development. Finally, a much larger than expected number of nominally intracellular proteins was detected. Most of these do not possess a signal sequence peptide. This suggests that nonclassical secretion plays a larger role than previously thought for many of the proteins identified. The secretory mechanisms by which some of the identified proteins are released into the extracellular milieu were investigated. Brefeldin A (BFA), an inhibitor of vesicular transport between the ER and Golgi apparatus, was used to determine whether identified proteins are secreted by a classical or non-classical secretory pathway. In addition, a preliminary study of an exosomal preparation derived from the secreted fraction of the same cell line was also undertaken. Many of the proteins identified in the exosomal pellet had also been detected in the secreted fractions, giving strong evidence that exosome-mediated secretion may represent one of the unconventional export mechanisms, for release of some of the identified proteins into the extracellular medium.