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. Pathophysiological 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 subtyping to prevent unnecessary adjuvant chemotherapy and to facilitate development of more effective therapies.2,3 Eukaryotic cell proliferation is normally an exquisitely choreographed 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 * To whom correspondence should be addressed. CH Best Institute, 112 College Street, Rm 402, Toronto, Ontario, Canada, M5G 1L6. Tel: (416) 9467281. Fax: (416) 978-8528. E-mail:
[email protected]. † University of Toronto. ‡ Sunnybrook Health Science Centre. § University of Miami.
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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 p27KIP1 correlate with aggressive malignancy and poor prognosis.12 Such differences have been attributed to epigenetic mechanisms, including promoter hypermethylation, 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 highprofile anti-Her-2 antibody therapy18) potently enhances mitogen-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 10.1021/pr0498842 CCC: $30.25
2005 American Chemical Society
Proliferating MCF-7 Breast Cancer Cells
to the characteristic uncontrolled proliferation that is a hallmark of malignancy.20 In contrast, in cervical cancers, oncogenesis is etiologically linked to infections by human papillomaviruses and is presumably initiated by targeted inactivation of select tumor suppressor proteins, such as p53 and pRb, by virally encoded oncoproteins.21 The advent of DNA microarray technology has allowed investigators to uncover global alterations in gene expression associated with neoplasia.22 Large-scale changes in mRNA levels are commonly seen in breast tumors, including marked alterations in the abundance of transcripts encoding proteins involved in the control of cell division.2,23 While many of the signaling pathways linked to breast cancer ultimately impinge on gene expression, protein levels can also be impacted in tumors by altered rates of translation and proteolysis. Moreover, it is now well established that mRNA levels do not always correlate with protein abundance.24 Impaired post-transcriptional regulation of key cell cycle regulators, such as p27KIP1 is commonly observed in breast cancer in the absence of detectable changes in transcript levels.25-27 These observations indicate the importance of directly assessing protein expression patterns associated with the breast cancer phenotype. Nevertheless, relatively few comprehensive proteomic investigations of breast cancer have been reported to date.28 Gel-free shotgun protein profiling procedures based on highresolution separation of tryptic peptide digests using capillaryscale multidimensional liquid chromatography coupled by electrospray ionization to ultra-sensitive micro-sequencing by tandem mass spectrometry represents a particularly powerful proteomic method for analyzing and comparing complex protein mixtures.29,30 By eliminating the need to separate proteins on polyacrylamide gels, this form of proteome mapping circumvents many of the limitations and biases associated with traditional gel-based screening procedures.30,31 Here, we report the results of a systematic, large-scale gelfree shotgun proteomic investigation of the global patterns of protein expression in proliferating MCF-7 cells in comparison to normal finite lifespan human mammary epithelial cells and to growth arrested (quiescent) cancer cell populations. Application of pattern recognition data-mining algorithms to the over 3500 proteins identified with high confidence revealed functionally related clusters whose expression patterns correlated closely with the breast cancer malignant phenotype. Combined with the results of recently reported microarray analyses of cancer gene expression profiles,2,3 this study provides an overview of the molecular alterations associated with breast cancer cell proliferation, and thereby offers a holistic framework for understanding the biochemical consequences of oncogenic transformation on normal breast epithelium.
Materials and Methods Cell Culture. Estrogen-sensitive MCF-7 cells were passaged in improved modified essential medium (IMEM-option Zn2+; GIBCO-BRL, Burlington, ON) supplemented with 5% fetal calf serum and insulin. MCF-7 cultures were arrested in the G1 phase by transferal to phenol red-free medium supplemented with 0.1% charcoal-stripped fetal bovine serum for 48 h. Quiescent cells were released into the cell cycle by the addition of 5% fetal bovine serum (GIBCO-BRL), insulin and 10-8 M estradiol (Sigma-Aldrich, St. Louis, MO). Cells (approximately 2 × 107) were harvested before (0 h; t ) 0) and 6, 12, 18, and 24 h after addition of serum and frozen prior to analysis.
research articles Normal control human mammary epithelial cells (HMEC cell line 184) derived from reduction mammoplasty, which can proliferate for up to 80 population doublings prior to undergoing senescence32 were a kind gift from Martha Stampfer (University of California, Lawrence Berkeley National Laboratory, CA). Experiments were performed using passage 12-14 cells cultured essentially as described.33 Cell Cycle Flow Cytometric Analysis. Cell aliquots were harvested by low speed centrifugation, washed with Phosphate Buffered Saline (PBS) and fixed with 70% ethanol. Fixed cells were treated with 0.1 N HCl and heated for 10 min at 95 °C to remove RNA, and cellular DNA then labeled with propidium iodide as described.34 Flow cytometry was carried out using a FACScan instrument (Becton Dickinson; Mississauga, ON) and the associated CellQuest software package. Protein Extract Preparation. Nuclear-enriched cell extracts were prepared using a commercial protocol (Nu-CLEAR protein extraction kit; Sigma-Aldrich) according to the manufacturer’s directions. Briefly, cell pellets were thawed, re-suspended in 5 volumes of hypotonic lysis buffer (10 mM HEPES, pH 7.9, 1.5 mM MgCl2, 10 mM KCl), and incubated on ice for 15 min. After re-pelleting by centrifugation for 5 min at 420 × g, the cells were washed twice in 400 µL of lysis buffer, re-suspended in high salt extraction buffer (1 M HEPES pH 7.9, 1 M MgCl2, 5 M NaCl, 0.5 M EDTA pH 8.0, 25% (vol/vol) glycerol), and incubated on ice for 15 min with occasional vigorous vortexing prior to the addition of the detergent NP40 to a final concentration of 0.04% (vol/vol). The cells were then disrupted by quick strokes with a glass homogenizer, followed by centrifugation for 5 min at 21 000 × g to remove debris. The soluble supernatant (final protein concentration ∼2-4 mg/mL) was saved for further analysis. For Western blotting, 50 µg total protein per lane was resolved by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE), transferred to nitrocellulose and probed as described previously35 using commercial antibodies to RCC1 and β-tubulin (Transduction Laboratories, Mississauga, ON; and Sigma-Aldrich, respectively). Protein Digestion. Aliquots (∼60 µg total protein) of nuclear extract were precipitated with 5 volumes of ice-cold acetone overnight at -20 °C. After pelleting by centrifugation for 20 min at 21 000 g, the protein was resolubilized in 8M urea, 50 mM Tris-HCl, pH 8.5, incubated for 2 h at 37 °C, and then reduced with 1 mM DTT followed by carboxyamidomethylation using 5 mM iodoacetamide. After dilution to [4 M] urea with 50 mM ammonium bicarbonate (AmBic), pH 8.5, the samples were digested with (1:50 w/w) endoproteinase Lys-C (Roche Diagnostics, Laval, QC) overnight at 37 °C. The next day, the samples were further diluted to [2 M] urea with AmBic, supplemented with CaCl2 to a final concentration of 1 mM, and extensively digested with Poroszyme immobilized trypsin (Applied Biosystems; Streetsville, ON) for 48 h at 30 °C with rotation. The resulting peptide mixtures were solid-phase extracted with SPEC-Plus PT C18 cartridges (Ansys Diagnostics; Lake Forest, CA) according to the manufactures instructions, and stored at -80 °C until further use. Shotgun Tandem Mass Spectrometry. Shotgun sequencing was performed using the multidimensional protein identification technology (MudPIT) developed by Yates and colleagues,29 as described by Kislinger et al.31 Briefly, a quaternary HPLC pump was interfaced using the electrospray ionization method to either an LTQ linear ion trap (for the MCF-7 versus 184 HMEC repeat analyses; see the Results) or an LCQ Deca XP Journal of Proteome Research • Vol. 4, No. 3, 2005 675
research articles quadrupole ion trap (for the MCF-7 time-course analysis) tandem mass spectrometers (Thermo Finnigan; San Jose, CA). The peptide samples were loaded onto separate 150 µm i.d. fused silica capillary micro-columns (Polymicro Technologies; Phoenix, AZ), each pulled to a fine tip on one end using a P-2000 laser puller (Sutter Instruments; Novato, CA) and packed with 10 cm of 5 µm Zorbax Eclipse XDB-C18 resin (Agilent Technologies, Mississauga, ON, Canada) followed by with 6 cm of 5 µm Partisphere strong ion exchange (SCX; Whatman, Clifton, NJ). For the MCF-7 versus 184 HMEC comparisons, each peptide mixture was loaded and analyzed separately in triplicate using 20 µg digested protein per analysis, whereas 100 µg of total protein digest was analyzed per time-point in the MCF-7 time-course study. Samples were loaded manually using a pressure vessel and analyzed via a fully automated four solvent, 12-step, 24-hour chromatographic cycle. MS/MS acquisition was performed in a data-dependent manner by operating the ion trap instrument using dynamic-exclusion lists. A detailed description of the chromatographic conditions is provided in Supporting Information. Database Searching. Uninterpreted peptide spectra were searched against a locally maintained minimally redundant database of human and mouse protein sequences (Swiss Protein and TrEMBL; obtained ca. August, 2003 from the European Bioinformatics Institute) using a distributed version of the SEQUEST algorithm36 running on a multi-processor computer cluster. To assess the False Discovery Rate, the sequences were present in both the normal and in a reversed (inverted) amino acid orientation,31 with matches to reversed proteins considered as false positives. Individual high-confidence protein matches were filtered using the STATQUEST probability assessment algorithm;31 a threshold minimum probability of 85% predicted likelihood in one ore more samples was used as cutoff. All LTQ-derived protein matches were additionally filtered based on a minimum cumulative spectral count of two. Hypothetical proteins detected in the time-course analysis were re-searched against the Swiss-Prot database using the BLAST algorithm to identify proteins bearing significant homology. Putative paralogs were identified with an E-value score of equal to or less than 10-30. Functional Annotation. High confidence proteins were classified and sorted into select Gene Ontology (GO) functional annotation categories37 using the GOClust program.31 Roughly half (53%) of the proteins could be linked to one or more GO terms. Closely related GO terms were manually combined to form the following larger super-GO categories: Cell Cycle (consisting of the GO terms cell cycle, cell cycle control, DNA replication, M phase and mitotic cell cycle), Stress/Death/ Apoptosis (stress response, death, and apoptosis regulators), Signal Transduction (signal transduction and phosphate metabolism), Transcriptional Regulation (transcriptional regulation), Catabolism and Energy pathways (catabolism and energy pathways), Translational Regulators (translational regulators and biosynthesis) and Protein Metabolism (protein metabolism and modification, regulation of metabolism and secondary metabolism). Hierarchical Clustering, Data Visualization and Cluster Evaluation. As recently noted by Yates and co-workers,38 cumulative spectral counts was used as a useful semiquantitative measure of relative protein abundance. For the timecourse analysis, relative protein levels were estimated using the ratio of the natural logarithm of spectral counts detected for each identified protein per time-point relative to that detected 676
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in the arrested (t ) 0) cell population as reference. Relative protein abundance in MCF-7 as compared to control 184 epithelial cells was calculated based on the ratio of median spectral count per protein. To detect statistically significant enrichment or depletion in GO functional categories, proteins exhibiting median ratios >2 or 2-fold) in MCF-7 relative to 184 HMEC, while 846 exhibited decreased ()>2-fold) expression in the cancer cells. To assess the functional significance of these putative global differences and to mitigate against over-interpretation of random spurious perturbations in the levels of a few select proteins, the overall memberships of the up- and down-regulated groups of proteins were examined separately for statistically significant (two sided p-value
