Plasma membrane proteomics of human breast cancer cell lines identifies potential targets for breast cancer diagnosis and treatment

Abstract

The use of broad spectrum chemotherapeutic agents to treat breast cancer results in substantial and debilitating side effects, necessitating the development of targeted therapies to limit tumor proliferation and prevent metastasis. In recent years, the list of approved targeted therapies has expanded, and it includes both monoclonal antibodies and small molecule inhibitors that interfere with key proteins involved in the uncontrolled growth and migration of cancer cells. The targeting of plasma membrane proteins has been most successful to date, and this is reflected in the large representation of these proteins as targets of newer therapies. In view of these facts, experiments were designed to investigate the plasma membrane proteome of a variety of human breast cancer cell lines representing hormone-responsive, ErbB2 over-expressing and triple negative cell types, as well as a benign control. Plasma membranes were isolated by using an aqueous two-phase system, and the resulting proteins were subjected to mass spectrometry analysis. Overall, each of the cell lines expressed some unique proteins, and a number of proteins were expressed in multiple cell lines, but in patterns that did not always follow traditional clinical definitions of breast cancer type. From our data, it can be deduced that most cancer cells possess multiple strategies to promote uncontrolled growth, reflected in aberrant expression of tyrosine kinases, cellular adhesion molecules, and structural proteins. Our data set provides a very rich and complex picture of plasma membrane proteins present on breast cancer cells, and the sorting and categorizing of this data provides interesting insights into the biology, classification, and potential treatment of this prevalent and debilitating disease.

Figure 1. Workflow outlines process of obtaining plasma membrane proteome of multiple BC cell lines.

After establishing an effective method for isolating plasma membrane proteins, representatives from each of the currently defined classes of BC were cultured and plasma membranes were isolated and subjected to MS, yielding a comprehensive list of protein identifications having an FDR ≤1%. These data were biologically validated and the data were mined for relevant protein candidates.

Figure 2. Western blots illustrate the purification of plasma membrane proteins using an aqueous two-phase system.

SK-BR-3 plasma membranes were isolated, and the input (IN), crude nuclear pellet (CNP) which also contained unlysed whole cells, cytosol (CYT), total cellular membranes (TM) which included nuclear, mitochondrial, endoplasmic reticular, and plasma membranes, and purified plasma membranes (PM) were subjected to Western blot analysis. ErbB2 is plasma membrane based, prohibitin is anchored in mitochondrial membranes, lamin A/C is found in nuclear membranes, and calnexin is an endoplasmic reticulum protein. All lanes were loaded at 10 µg protein/lane.

Figure 3. Comparison of PM proteomes shows similarities and differences among the cell lines.

A. Relationships between the data sets can be visualized by clustering those that share similar expression profiles. B. Venn diagrams highlight the degree of similarity between DT22 and MDA-MB-231 (231) cells compared to DT22 and MCF-10A (10A) cells.

Figure 4. RT-PCR demonstrates the quantitative nature of the MS data.

RNA was isolated from each of the cell lines, cDNA was made, and RT-PCR was performed to determine whether the spectral ID numbers were correlated to transcript levels of selected genes. Spectral ID numbers are displayed above each graph and the gene symbol is below those numbers.

Figure 5. Western blots and immunofluorescence demonstrate that protein levels are accurately reflected in the MS data.

A. For Western analysis, PMs were isolated from each of the cell lines, solubilized in detergent buffer, and fractionated on SDS-PAGE gels. Blots were probed with an ErbB2- or KRT17-specific antibody and imaged with the Licor Infrared Imaging System. Spectral ID numbers and cell lines are indicated for each lane on the blot. B. For immunofluorescence, cells were grown in chamber slides, treated with an antibody to ErbB2 or KRT17 (red), and cell nuclei were stained with DAPI (blue). Cells were examined with confocal microscopy. All scale bars  = 25 µ.