Global profiling of signaling networks: study of breast cancer stem cells and potential regulation.

There is overwhelming evidence that breast cancer may be driven by a small subset of breast cancer stem cells (BCSCs) that display stem/progenitor cell properties. In the present study, we identified the rare population of BCSCs, the so-called side population (SP) cells, using flow cytometry. Then, we used microarray analysis to study the differential gene expression profiles between SP and non-SP cells. Sixty-three probe sets showed a more than fourfold difference. Next, we compared the levels of proteins with Pathway Array using 154 antibodies, focusing on the proteins and phosphorylation sites that differed among SP cells, malignant mammary cells, and breast cancer tissues. Our results revealed that 40 proteins and phosphorylation sites were more than 1.5-fold different in SP cells than in non-SP cells. By comparing SP cells, MCF7 cells, and nontumorigenic MCF10A cells, we found 12 proteins that were significantly upregulated in SP cells; these proteins-cAMP-response element binding protein (CREB), cyclic AMP-dependent transcription factor 1, mesothelin, thyroid transcription factor 1, phosphorylated (p)-focal adhesion kinase, p38, Bad, p-CREB, p-protein kinase C (PKC)δ, Wee1, cell division cycle 42, and Twist-were more likely to play important roles in the signaling regulation of BCSCs. Further, 16 proteins and phosphoproteins showed differential expression in SP cells and tumor tissues. β-catenin, p-PKCα, and p-CREB were upregulated in both SP cells and breast tumors. Finally, we filtered the differential expression proteins, summarized the pathway interactions of these proteins, and rebuilt Path-Net in order to determine molecular mechanisms and core regulators. This process will allow us to identify signature patterns and mechanisms of signaling networks in BCSCs.