Global Protein Shotgun Expression Profiling of Proliferating MCF-7 Breast Cancer Cells Charanjit Sandhu, † Michael Connor, ‡ Thomas Kislinger, † Joyce Slingerland, § and Andrew Emili* ,† Program in Proteomics and Bioinformatics, Banting and Best Department of Medical Research, University of Toronto, Toronto, Ontario, Canada, Division of Cancer Biology Research, Sunnybrook Health Science Centre, Toronto, Ontario, Canada, and Braman Breast Cancer Institute, Sylvester Comprehensive Cancer Center, University of Miami, Miami, Florida Received July 13, 2004 Protein expression becomes altered in breast epithelium during malignant transformation. Knowledge of these perturbations should provide insight into the molecular basis of breast cancer, as well as reveal possible new therapeutic targets. To this end, we have performed an extensive comparative proteomic survey of global protein expression patterns in proliferating MCF-7 breast cancer cells and normal human mammary epithelial cells using gel-free shotgun tandem mass spectrometry. Patho- physiological alterations associated with the malignant breast cancer phenotype were detected, including differences in the apparent levels of key regulators of the cell cycle, signal transduction, apoptosis, transcriptional regulation, and cell metabolism. Keywords: mass spectrometry • protein expression profiling • proteomics • breast cancer • cell cycle • proliferation Introduction Breast cancer is the most frequently diagnosed cancer in women in the developed world, with over 190 000 new cases reported each year in the US alone, and is the second leading cause of cancer-associated mortality in females. 1 Despite recent advances, clinical treatment of breast cancer still suffers from a paucity of validated prognostic tools to identify patients at high risk of dying or being nonresponsive to therapy. 2 There is therefore a pressing need to identify biochemical markers associated with malignant growth that could aid in disease sub- typing to prevent unnecessary adjuvant chemotherapy and to facilitate development of more effective therapies. 2,3 Eukaryotic cell proliferation is normally an exquisitely cho- reographed molecular performance executed by the mitogens that stimulate cell growth, the receptors and signaling pathways on which they act, and the downstream mediators and effectors of cell division. Proper cell cycle progression consists of an ordered series of biochemical transitions, such as the initiation of DNA replication and mitotic chromosome disjunction, which are closely coordinated with cell growth. 4 Accurate control of these events is essential for genomic stability. 5 Neoplastic transformation, in turn, is characterized by aberrations in the synthesis, accumulation or degradation of regulatory proteins that modulate cell growth and division. 6,7 Identification of critical cell cycle-regulated proteins whose levels become altered in breast cancer is providing insight into the molecular mechanisms that govern cancer cell proliferation. 6,7 Although inactivation of the BRCA1 and BRCA2 DNA repair proteins has been linked to familial forms of breast cancer, 8 much remains to be learned about the biochemical adaptations associated with sporadic forms of the disease. Cell cycle-related perturbations are ubiquitous in breast cancer. 9 These include loss of critical tumor suppressors, and overexpression of growth-stimulating oncoproteins. 10,11 For instance, low levels of p16, an inhibitor of cyclin-dependent kinases (CDKs) required for entry into S-phase, and elevated expression of cyclin D1 correlate with slow tumor growth and long-term survival, whereas elevated cyclin E and low levels of the CDK inhibitor p27 KIP1 correlate with aggressive malignancy and poor prognosis. 12 Such differences have been attributed to epigenetic mechanisms, including promoter hypermethy- lation, differential post-translation modification and aberrant protein turnover. 13-15 The estrogen-dependent MCF-7 malignant breast epithelial cell line is a well-studied model system of breast cancer cell transformation and proliferation, 16,17 including investigation of the effects of estrogen. Estrogen binding to the ER receptor potently stimulates MCF-7 mitogenic pathways, oncogenic initiation and progression. 17 ER-dependent (ER+) breast tumors exhibit slower growth and improved prognostic outcome, 17 whereas expression of the Her-2/neu receptor (target of high- profile anti-Her-2 antibody therapy 18 ) potently enhances mi- togen-mediated MCF-7 cell growth and correlates strongly with poor disease outcome. 19 These and other studies suggest that oncoplasmic transformation involves synergistic deregulation of multiple signaling pathways and cell cycle control, leading * To whom correspondence should be addressed. CH Best Institute, 112 College Street, Rm 402, Toronto, Ontario, Canada, M5G 1L6. Tel: (416) 946- 7281. Fax: (416) 978-8528. E-mail: andrew.emili@utoronto.ca. † University of Toronto. ‡ Sunnybrook Health Science Centre. § University of Miami. 674 Journal of Proteome Research 2005, 4, 674-689 10.1021/pr0498842 CCC: $30.25  2005 American Chemical Society Published on Web 05/20/2005