Patent Application
20110251082
The present invention relates to biomarkers for breast cancer. More specifically, the invention relates to biomarkers that can be used in diagnosis, determination of disease severity, and monitoring of therapeutic response of patients with breast cancer. The method is based on the use of 2-dimensional differential gel electrophoresisgel (2D-DIGE) to quantitatively identify biomarkers in breast cancer.
Breast cancer is one of the leading causes of death among women around the world. The 5-year survival rate for breast cancer is close to 97% when tumors are confined to breast tissue, but decrease dramatically to 23% when tumors have metastasized to other organs at the time of diagnosis. Presymtomatic screening to detect early-stage breast cancer while it is still resectable with potential for cure can greatly reduce breast cancer-related mortality. Unfortunately, only 63% (1992-1999, US) of the breast cancers are localized at the time of diagnosis (Jemal, A. et al. (2004) CA Cancer J. Clin. 54:8-29). Small lesions are frequently missed and may not be visible, even by mammography, particularly in young women and women with dense breast tissue. Molecular markers that can potentially identify these small lesions that are invisible to imaging techniques will provide a real opportunity to treat a neoplasm before it invades tissue.
Previous inventions indicate that the transformation and metastasis of normal breast cells are correlated to altered expression in both transcription and translation levels (Kulasingam, V. & Diamandis, E. P. Mol. Cell. Proteomics 2007, 6, 1997). To better understand the molecular mechanisms associated with tumorigenesis and metastasis, it is necessary to identify the gene expression signatures and protein expression markers among normal breast cells, noninvasive breast cancer cells, and invasive breast cancer cells. At the transcription level, microarray strategies have been used to classify breast tumors as highly invasive and noninvasive cancer. At the translation level, proteomic strategies have been used to discern cancer markers from noninvasive and invasive breast cells. Nagaraja et al. compared the proteomic profiling of cell lines corresponding to normal breast cells, non-invasive breast cancer cells, and invasive breast cancer cells using 2-DE (Nagaraja, G. M.; Othman, M.; Fox, B. P.; Alsaber, R.; Pellegrino, C. M.; Zeng, Y.; Khanna, R.; Tamburini, P.; Swaroop, A.; Kandpal, R. P. Oncogene 2006, 25, 2328). Although they identified 26 spots as potential cancer markers, no statistical analysis was included in their study. Pucci-Minafra et al. compared a ductal infiltrating carcinoma-derived cell line with a non-tumoral mammary epithelial cell line using 2-DE, silver staining, immunodetection, and N-terminal sequencing and identified 58 differentially expressed proteins (Pucci-Minafra, I.; Fontana, S.; Cancemi, P.; Alaimo, G; Minafra, S. Ann. N.Y. Acad. Sci. 2002, 963, 122). In contrast to these cell line based experiments, Pawlik analyzed differentially expressed proteins among nipple aspirate fluid samples from tumor-bearing and disease-free breasts (Pawlik, T. M.; Hawke, D. H.; Liu, Y.; Krishnamurthy, S.; Fritsche, H.; Hunt, K. K.; Kuerer, H. M. BMC. Cancer 2006, 6, 68). Although these identified proteins are primarily abundant proteins, few of them have been validated as biomarkers.
The present invention provides a breast cancer biomarker library, comprising bestrophin-3, carbonic anhydrase 2, dynein heavy chain 6, ecto-ADP-ribosyltransferase 4, GRAM domain-containing protein 2, interferon-induced protein with tetratricopeptide repeat 3, phosphoglycerate mutase 1, proteasome subunit alpha type-1, proteasome subunit alpha type-3, rab GTPase-binding effector protein 2, Ras-related protein Rab-2B, selenium-binding protein 1, transmembrane protein C14orf180, vascular protein sorting-associated protein 54, achaete-scute homologue 4, aconitate hydratase, aminopeptidase B, annexin A3, barrier-to-autointegration factor, bifunctional purine biosynthesis, calumenin, carbonic anhydrase 2, coiled-coil domain-containing protein, erlin-2, F-actin-capping protein subunit beta, flavin reductase, fructose-1,6-biphosphatase 1, fructose-biphosphate ldolase A, heat shock protein 75 kDa, heterogeneous nuclear ribonucleoproteins A2/B1, leukotriene A-4 hydrolase, microfibrillar-associated protein 3 like, microtubule-associated protein RP, nuclear distribution protein nudE homologue 1, parvalbumin alpha, PDZ and LIM domain protein 1, peptidylprolyl isomerase domain and WD repeat-containing protein 1, phosphoserine aminotransferase, plastin-3, programmed cell death 6-interacting protein, proteasome activator complex subunit 1, proteasome activator complex subunit 2, protein canopy homologue 2, protein CASC2, protein disulfide-isomerase A6, protein SHQ1, Rab GDP dissociation inhibitor beta, reticulocalbin-2, Rho GTPase-activating protein 25, Rho GTPase-activating protein 5, ribonuclease inhibitor, selenium-binding protein 1, septin-11, septin-8, serine/threonine-protein kinase Nek7, serine/threonine-protein kinase PCTAIRE-1, small ubiqutin-related modifier 3, stress-induced phosphoprotein 1, thioredoxin domain-containing protein 5, ubiquitin-conjugating enzyme E2, UPF0492 protein C20orf94, voltage-dependent anion-selective channel protein and zinc finger protein 433.
The present invention also provides a method of predicting an increased likelihood of developing breast cancer progression of a subject comprising:
The present invention provides a biomarker library for breast cancer, and a method of predicting an increased likelihood of developing breast cancer progression using the same.
Breast cancer is the leading cause of cancer-induced mortality in women. Early detection of breast cancer greatly improves its survival rates. The identification of cellular targets that play a role in highly invasive breast cancer may also contribute to a better understanding of the biological mechanisms inherent in the aggressive progression of cancer and may also be used in the development of new diagnostic or therapeutic strategies for breast cancer.
Accordingly, the aim of the present invention is to discover biomarkers with the greatest potential to facilitate early detection of breast cancer and monitor the progress of breast tumorigenesis. Numerous proteins, including bestrophin-3 and parvalbumin, are highly expressed in both low-invasive and aggressive breast cancer cells and are verified as breast cancer markers in this invention. Importantly, several of these identified proteins, including bestrophin-3, GRAMD2, and nuclear distribution protein nudE homolog 1, have not been reported in previous breast cancer studies, thus implying these proteins are valuable breast cancer markers.
Biomarkers of the present invention can be used in diagnosis, including determination of disease severity and monitoring of therapeutic response of patients with breast cancer. The biomarker library is listed as follows: bestrophin-3, carbonic anhydrase 2, dynein heavy chain 6, ecto-ADP-ribosyltransferase 4, GRAM domain-containing protein 2, interferon-induced protein with tetratricopeptide repeat 3, phosphoglycerate mutase 1, proteasome subunit alpha type-1, proteasome subunit alpha type-3, rab GTPase-binding effector protein 2, Ras-related protein Rab-2B, selenium-binding protein 1, transmembrane protein C14orf180, vascular protein sorting-associated protein 54, achaete-scute homologue 4, aconitate hydratase, aminopeptidase B, annexin A3, barrier-to-autointegration factor, bifunctional purine biosynthesis, calumenin, carbonic anhydrase 2, coiled-coil domain-containing protein, erlin-2, F-actin-capping protein subunit beta, flavin reductase, fructose-1,6-biphosphatase 1, fructose-biphosphate ldolase A, heat shock protein 75 kDa, heterogeneous nuclear ribonucleoproteins A2/B1, leukotriene A-4 hydrolase, microfibrillar-associated protein 3 like, microtubule-associated protein RP, nuclear distribution protein nudE homologue 1, parvalbumin alpha, PDZ and LIM domain protein 1, peptidylprolyl isomerase domain and WD repeat-containing protein 1, phosphoserine aminotransferase, plastin-3, programmed cell death 6-interacting protein, proteasome activator complex subunit 1, proteasome activator complex subunit 2, protein canopy homologue 2, protein CASC2, protein disulfide-isomerase A6, protein SHQ1, Rab GDP dissociation inhibitor beta, reticulocalbin-2, Rho GTPase-activating protein 25, Rho GTPase-activating protein 5, ribonuclease inhibitor, selenium-binding protein 1, septin-11, septin-8, serine/threonine-protein kinase Nek7, serine/threonine-protein kinase PCTAIRE-1, small ubiqutin-related modifier 3, stress-induced phosphoprotein 1, thioredoxin domain-containing protein 5, ubiquitin-conjugating enzyme E2, UPF0492 protein C20orf94, voltage-dependent anion-selective channel protein and zinc finger protein 433.
When using biomarkers in diagnosis, the present invention provides a direct way of predicting an increased likelihood of developing breast cancer progression of a subject comprising following steps:
The expression pattern indicates the quantity of biomarker expression. In a better embodiment, the increasing or decreasing of biomarker expression is 1.5 fold relative to the biomarker expression of normal tissue.
The method further comprises applying software for protein expression comparison between normal tissue and tumor tissue.
The breast cancer progression includes the presence or absence of breast tumor, the stage of breast cancer and the effectiveness of breast cancer treatment.
The stage of breast cancer includes invasive and non-invasive tumor pregression. Invasive tumor is another synonym of cancer, and the name refers to invasion of surrounding tissues. Non-invasive tumor is a neoplasm which is not invasive but has the potential to progress to cancer (become invasive) if left untreated.
A neoplasm is the abnormal proliferation of cells. The growth of this clone of cells exceeds, and is uncoordinated with, that of the normal tissues around it. The growth persists in the same excessive manner even after cessation of the stimuli. It usually causes a lump or tumor.
The subject mentioned herein is human or mammal, and the sample is selected from blood, serum, plasma, ductal lavage fluid and nipple aspiration fluid.
The bioassay used herein comprises immunoassay, electrophoresis and mass spectrum.
The immunoassay is measured using an immunoblotting, especially an antibody-based assay. The antibody-based assay used herein is for biomarker detection, comprsing at least one vessel for antibody and biomarker interaction and a detectable label to attached with antibody. Useful detectable labels include but are not limited to radioactive labels such as 32P, 3H, and 14C; fluorescent dyes such as fluorescein isothiocyanate (FITC), rhodamine, lanthanide phosphors, Texas red, and ALEXA Fluor Dyes™ (Molecular Probes), CY™ dyes (Amersham), Spectrum Dyes (Abbott Labs); electron-dense reagents such as gold; enzymes such as horseradish peroxidase, beta-galactosidase, luciferase, and alkaline phosphatase; colorimetric labels such as colloidal gold; magnetic labels such as those sold under the mark DYNABEADS™; biotin; dioxigenin; or haptens and proteins for which antisera or monoclonal antibodies are available. The label can be directly incorporated into the polynucleotide, or it can be attached to a molecule which hybridizes or binds to the polynucleotide. The labels may be coupled to the isolated polynucleotides by any means known to those of skill in the art. In various embodiments, the isolated polynucleotides are labeled using nick translation, PCR, or random primer extension (see, e.g., Sambrook et al. supra). Methods for detecting the label include, but are not limited to spectroscopic, photochemical, biochemical, immunochemical, physical or chemical techniques.
The electrophoresis used herein is 2-dimensional differential gel electrophoresisgel (2D-DIGE), which works efficiently to identify the quantity of breast cancer biomarker expression.
2-DE is currently a key technique in profiling thousands of proteins within biological samples and plays a role complementary to LC/MS-based proteomic analysis. However, reliable quantitative comparisons between gels and gel-to-gel variations remain the primary challenge in 2-DE analysis. A significant improvement in the gel-based analysis of protein quantitation and detection was achieved by the introduction of 2D-DIGE, which can co-detect numerous samples in the same 2-DE. This approach minimizes gel-to-gel variations and compares the relative amount of protein features across different gels using an internal fluorescent standard. Moreover, the 2D-DIGE technique has the advantages of a broader dynamic range, higher sensitivity, and greater reproducibility than traditional 2-DE. This innovative technology relies on the pre-labeling of protein samples with fluorescent dyes (Cy2, Cy3 and Cy5) before electrophoresis. Each dye has a distinct fluorescent wavelength, allowing multiple experimental samples with an internal standard to be simultaneously separated in the same gel. The internal standard, which is a pool of an equal amount of the experimental protein samples, helps provide accurate normalization data and increase statistical confidence in relative quantitation among gels.
Whether or not comparisons of normal cell lines with cancer cell lines actually reflect common changes associated with cancer and can be successfully developed into clinically useful biomarkers or therapeutic targets remains debatable. Thus, a direct comparison of cancer tissue with normal tissue is the best theoretical method of obtaining protein expression signatures during tumor progression. However, a direct comparison of clinical samples increases the amount of false positives due to the heterogeneity of tumor specimens, which interferes with the identification of tumor-specific markers. For this reason, well-characterized model cell lines established from normal and tumor tissue are recognized as more informative in cancer proteomics research.
In the field of breast cancer research, MCF-10A, MCF-7 and MDA-MB-231 are widely used to represent normal luminal epithelial cells, non-invasive breast cancer cells derived from the luminal duct and invasive breast cancer cells derived from the same tissue, respectively. The present invention compares the proteomic profiles of total cellular proteins and secreted proteins of this cell model system using 2D-DIGE to quantitatively identify biomarkers in breast cancer, wherein the biomarkers reflect the progression of tumorigenesis.
The results show differentially expressed protein profiles across normal and transformed breast cell lines, ranging from extracellular secreted proteins and intracellular proteins. The 2D-DIGE strategy is powerful enough to identify numerous breast cancer signatures and offers a complementary role to LC/MS-based proteomic analysis. Even though the global coverage of protein mixtures identified by LC-MS based analysis is generally higher than that of 2-DE based analysis, 2-DE based analysis offers some distinct advantages, such as direct protein quantification at protein isoform levels instead of peptide levels to reduce analytical variations.
The examples below are non-limiting but merely representative of various aspects and features of the present invention.
Generic chemicals were purchased from Sigma-Aldrich (St. Louis, USA), while reagents for 2D-DIGE were purchased from GE Healthcare (Uppsala, Sweden). All primary antibodies were purchased from Abcam (Cambridge, UK) and anti-mouse, anti-goat and anti-rabbit secondary antibodies were purchased from GE Healthcare (Uppsala, Sweden). All chemicals and biochemicals used in the present invention were of analytical grade.
The breast epithelial cell line MCF-10A was from National Health Research Institute, Taiwan. The breast cancer cell lines MCF-7, MDA-MB-231, MDA-MB-453 and MDA-MB-361 were purchased from American Type Culture Collection (ATCC), Manassas, Va. MCF-10A was maintained in Dulbecco's Modified Eagle's medium and F-12 medium (DMEM/F-12) supplemented with 5% horse serum, L-glutamine (2 mM), streptomycin (100 μg/mL), penicillin (100 IU/mL), epidermal growth factor (20 ng/ml) (all from Gibco-Invitrogen Corp., UK), insulin (10 μg/ml) (Sigma) and hydrocortisone (0.5 μg/ml) (Sigma). MCF-7, MDA-MB-231, MDA-MB-453 and MDA-MB-361 were maintained in Dulbecco's Modified Eagle's medium (DMEM) supplemented with 10% (v/v) fetal calf serum (FCS), L-glutamine (2 mM), streptomycin (100 μg/mL), and penicillin (100 IU/mL) (all from Gibco-Invitrogen Corp., UK). All cells were incubated at 37° C. and 5% CO2.
Cells in normal growth medium at ˜80% confluence were used for proteomic analysis. For total cellular protein analysis, cells were washed in chilled 0.5×PBS and scraped in 2-DE lysis buffer containing 4% w/v CHAPS, 7M urea, 2M thiourea, 10 mM Tris-HCl, pH8.3, 1 mM EDTA. Lysates were homogenized by passage through a 25-gauge needle 10 times, insoluble material was removed by centrifugation at 13000 rpm for 30 min at 4° C., and protein concentrations were determined using Coomassie Protein Assay Reagent (BioRad). For secreted protein analysis, approximately 1.25×108 cells were seeded into twenty-five 175-cm2 cell culture plates for each cell line. After 2 days of incubation, the DMEM or DMEM/F-12 media were discarded, and the cells were rinsed three times with PBS. Subsequently, 375 ml of serum-free DMEM or DMEM/F-12 media were added for an additional 30 hours. The media were collected and filtered with 0.45 μm microfilters to remove cell debris and then concentrated 1000 fold with 10-kDa molecular mass cutoff concentrators (Millipore). The concentrated media were then precipitated by adding 1 volume of 100% TCA (at −20° C.) to 4 volumes of sample and incubated for 10 min. at 4° C. The precipitated protein was then recovered by centrifugation at 13000 rpm for 10 min., and the resulting pellet was washed twice with ice-cold acetone. Air-dried pellets were resuspended in 2-DE lysis buffer for protein quantification.
Before performing 2D-DIGE, protein samples were labeled with N-hydroxy succinimidyl ester-derivatives of the cyanine dyes Cy2, Cy3 and Cy5. Briefly, 150 μg of protein sample was minimally labeled with 375 pmol of either Cy3 or Cy5 for comparison on the same 2-DE. To facilitate image matching and cross-gel statistical comparison, a pool of all samples was also prepared and labeled with Cy2 at a molar ratio of 2.5 pmol Cy2 per μg of protein as an internal standard for all gels. Thus, the triplicate samples and the internal standard could be run and quantify on multiple 2-DE. The labeling reactions were performed in the dark on ice for 30 min and then quenched with a 20-fold molar ratio excess of free L-lysine to dye for 10 min. The differentially Cy3- and Cy5-labeled samples were then mixed with the Cy2-labeled internal standard and reduced with dithiothreitol for 10 min. IPG buffer, pH3-10 nonlinear (2% (v/v), GE Healthcare) was added and the final volume was adjusted to 450 μl with 2D-lysis buffer for rehydration. The rehydration process was performed with immobilized non-linear pH gradient (IPG) strips (pH3-10, 24 cm) which were later rehydrated by CyDye-labeled samples in the dark at room temperature overnight (at least 12 hours). Isoelectric focusing was then performed using a Multiphor II apparatus (GE Healthcare) for a total of 62.5 kV-h at 20° C. Strips were equilibrated in 6M urea, 30% (v/v) glycerol, 1% SDS (w/v), 100 mM Tris-HCl (pH8.8), 65 mM dithiothreitol for 15 min and then in the same buffer containing 240 mM iodoacetamide for another 15 min. The equilibrated IPG strips were transferred onto 26×20-cm 12.5% polyacrylamide gels casted between low fluorescent glass plates. The strips were overlaid with 0.5% (w/v) low melting point agarose in a running buffer containing bromophenol blue. The gels were run in an Ettan Twelve gel tank (GE Healthcare) at 4 Watt per gel at 10° C. until the dye front had completely run off the bottom of the gels. Afterward, the fluorescence 2-DE was scanned directly between the low fluorescent glass plates using an Ettan DIGE Imager (GE Healthcare). This imager is a charge-coupled device-based instrument that enables scanning at different wavelengths for Cy2-, Cy3-, and Cy5-labeled samples. Gel analysis was performed using DeCyder 2-D Differential Analysis Software v7.0 (GE Healthcare) to co-detect, normalize and quantify the protein features in the images. Features detected from non-protein sources (e.g. dust particles and dirty backgrounds) were filtered out. Spots displaying a ≧1.5 average-fold increase or decrease in abundance with a p-value <0.05 were selected for protein identification.
Colloidal coomassie blue G-250 staining was used to visualize CyDye-labeled protein features in 2-DE. Bonded gels were fixed in 30% v/v ethanol, 2% v/v phosphoric acid overnight, washed three times (30 min each) with ddH2O and then incubated in 34% v/v methanol, 17% w/v ammonium sulphate, 3% v/v phosphoric acid for 1 hr., prior to adding 0.5 g/liter coomassie blue G-250. The gels were then left to stain for 5-7 days. No destaining step was required. The stained gels were then imaged on an ImageScanner III densitometer (GE Healthcare).
Excised post-stained gel pieces were washed three times in 50% acetonitrile, dried in a SpeedVac for 20 min., reduced with 10 mM dithiothreitol in 5 mM ammonium bicarbonate pH 8.0 (Ammonium bicarbonate) for 45 minutes at 50° C. and then alkylated with 50 mM iodoacetamide in 5 mM Ammonium bicarbonate for 1 hr. at room temperature in the dark. The gel pieces were then washed three times in 50% acetonitrile and vacuum-dried before reswelling with 50 ng of modified trypsin (Promega) in 5 mM Ammonium bicarbonate. The pieces were then overlaid with 10 μl of 5 mM Ammonium bicarbonate and trypsinized for 16 hr at 37° C. Supernatants were collected, peptides were further extracted twice with 5% trifluoroacetic acid in 50% acetonitrile and the supernatants were pooled. Peptide extracts were vacuum-dried, resuspended in 5 μl ddH2O, and stored at −20° C. prior to MS analysis.
Extracted proteins were cleaved with a proteolytic enzyme to generate peptides, then a peptide mass fingerprinting (PMF) database search following MALDI TOF mass analysis was employed for protein identification. Briefly, 0.5 μl of tryptic digested protein sample was first mixed with 0.5 μl of a matrix solution containing α-cyano-4-hydroxycinammic acid at a concentration of 1 mg in 1 ml of 50% acetonitrile (v/v)/0.1% trifluoroacetic acid (v/v), spotted onto an anchorchip target plate (Bruker Daltonics) and dried. The peptide mass fingerprints were acquired using an Autoflex III mass spectrometer (Bruker Daltonics) in reflector mode. The algorithm used for spectrum annotation was SNAP (Sophisticated Numerical Annotation Procedure). This process used the following detailed metrics: Peak detection algorithm: SNAP; Signal to noise threshold: 25; Relative intensity threshold: 0%; Minimum intensity threshold: 0; Maximal number of peaks: 50; Quality factor threshold: 1000; SNAP average composition: Averaging; Baseline subtraction: Median; Flatness: 0.8; MedianLevel: 0.5. The spectrometer was also calibrated with a peptide calibration standard (Bruker Daltonics) and internal calibration was performed using trypsin autolysis peaks at m/z 842.51 and m/z 2211.10. Peaks in the mass range of m/z 800-3000 were used to generate a peptide mass fingerprint that was searched against the Swiss-Prot/TrEMBL database (v57.12) with 513877 entries using Mascot software v2.2.06 (Matrix Science, London, UK). The following parameters were used for the search: Homo sapiens; tryptic digest with a maximum of 1 missed cleavage; carbamidomethylation of cysteine, partial protein N-terminal acetylation, partial methionine oxidation and partial modification of glutamine to pyroglutamate and a mass tolerance of 50 ppm. Identification was accepted based on significant MASCOT Mowse scores (p<0.05), spectrum annotation and observed versus expected molecular weight and pI on 2-DE.
Immunoblotting was used to validate the differential expression of mass spectrometry identified proteins. Cells were lysed with a lysis buffer containing 50 mM HEPES pH 7.4, 150 mM NaCl, 1% NP40, 1 mM EDTA, 2 mM sodium orthovanadate, 100 μg/mL AEBSF, 17 μg/mL aprotinin, 1 μg/mL leupeptin, 1 μg/mL pepstatin, 5 μM fenvalerate, 5 μM BpVphen and 1 μM okadaic acid prior to protein quantification with Coomassie Protein Assay Reagent (BioRad).
30 μg of protein samples were diluted in Laemmli sample buffer (final concentrations: 50 mM Tris pH 6.8, 10% (v/v) glycerol, 2% SDS (w/v), 0.01% (w/v) bromophenol blue) and separated by 1D-SDS-PAGE following standard procedures. After electroblotting separated proteins onto 0.45 μm Immobilon P membranes (Millipore), the membranes were blocked with 5% w/v skim milk in TBST (50 mM Tris pH 8.0, 150 mM NaCl and 0.1% Tween-20 (v/v)) for 1 hour. Membranes were then incubated in primary antibody solution in TBS-T containing 0.02% (w/v) sodium azide for 2 hours. Membranes were washed in TBS-T (3minutes, 10 times) and then probed with the appropriate horseradish peroxidase-coupled secondary antibody (GE Healthcare). After further washing in TBS-T, immunoprobed proteins were visualized using an enhanced chemiluminescence method (Visual Protein Co.).
For immunofluorescence staining, cells were plated onto coverslips (VWR international) for overnight incubation. The cells were fixed with PBS containing 4% (v/v) paraformaldehyde for 25 minutes, washed three times with PBS, and followed by permeabilization in PBS containing 0.2% (v/v) Triton X-100 for 10 minutes. Coverslips were rinsed and blocked in PBS containing 5% (w/v) BSA for 10 minutes before incubation with primary antibodies diluted in 2.5% BSA/PBS for 1 hour. After three washings with PBS, samples were incubated with the appropriate fluorescently labelled secondary antibodies diluted in 2.5% BSA/PBS for 1 hour. Coverslips were then washed three times with PBS and at least twice with ddH2O before mounting in Vectashield mounting medium (Vector Lab). Coverslip edges were sealed with nail polish onto glass slides (BDH) and then dried in the dark at 4° C. For image analysis, cells were imaged using a Zeiss Axiovert 200M fluorescent microscope (Carl Zeiss Inc., Germany). The laser intensities used to detect the same immunostained markers from different cell lines were identical, and none of the laser intensities used to capture images was saturated.
For secretomic analysis, MCF-10A, MCF-7 and MDA-MB-231 were grown on cell culture dishes and the confluency of cells was checked prior to incubation in serum-free culture media to ensure that no other exogenous proteins were present. To minimize cell autolysis induced by starvation and to maximize secreted protein concentration in the media, the starvation time of each cell line was optimized. Through immunoblotting, the LDH and β-tubulin levels were detected in the 1000-fold concentrated serum-free media starting at 48˜60 hours and at 60˜72 hours, respectively (
Accordingly, a starvation period of 30 hours was chosen for further 2D-DIGE based secretomic analysis.
DIGE and MALDI-TOF Analysis of Secretomes among MCF-10A, MCF-7 and MDA-MB-231 Cells
Proteins secreted from each cell type were enriched from the serum-free medium followed by labeling with CyDyes for 2D-DIGE analysis. The secretomic profiling of MCF-10A, MCF-7 and MDA-MB-231 were visualized using a fluorescence scanner and the images were superimposed using ImageQuant software (
Using the LC-MS/MS strategy, Kulasingam and Diamandis analyzed and compared the expressions of extracellular and membrane-bound proteins in conditioned media of three breast cells corresponding to the normal control cells and cell lines derived from stage 2 and stage 4 patients, respectively (Kulasingam, V.; Diamandis, E. P. Mol. Cell Proteomics. 2007, 6, 1997). Kulasingam's experiment identified 1062 differentially expressed proteins across these three cell lines. A comparison between Kulasingam's result and 2D-DIGE secretomic of the present invention shows that 25 out of 50 identified differentially expressed secreted proteins coincide with Kulasingam's study, indicating that both LC-MS/MS and 2D-DIGE are potential tools for discovering breast cancer markers with reasonable reproducibility. Importantly, 25 out of 50 identified proteins were not reported in Kulasingam's study or any other studies, demonstrating that 2D-DIGE plays a powerful complementary role in the assumed biomarker discovery (Table 1a & 1b).
#The everage ratio of differentially expressed (p < 0.05) proteins after 2D-DIGE analysis across MCF-10A, MCF-7, and MDA-MB-231 were calculated considering 3 replica gels.
##Identified protiens which have not been reported in any cancer research are marked “A”, while proteins which have been reported in cancer research but not in breast cancer research are marked “B”.
###Poreins in this list have been reported in Kulasing et al's experiment.
To identify the altered abundances of proteins and relate them to the tumorigenesis of breast cancer, the proteomic profiles of MCF-10A, MCF-7 and MDA-MB-231 were analyzed. Triplicates of the three different cell lysates were compared using 2D-DIGE to obtain an overview of breast cell tumorigenesis. Image analysis using DeCyder v7.0 clearly defined more than 2500 protein spots (
Furthermore, biological variation analysis of spots showing greater than 1.5-fold change in expression with a t-test score of less than 0.05 were visually checked before confirming the alterations for protein identification. MALDI-TOF MS identification revealed 133 unique differentially expressed proteins across MCF-10A, MCF-7, and MDA-MB-231 (Table 2). Of the 133 proteins identified, 107 of them had differential expressions between MCF-7/MCF-10A, 63 were differentially expressed between MDA-MB-231/MCF-10A and 96 had differential expressions between MDA-MB-231 and MCF-7. Almost half of the total proteins identified in this breast cell model were cytosolic proteins (
#The everage ratio of differentially expressed (p < 0.05) proteins after 2D-DIGE analysis
##Identified protiens which have not been reported in any cancer research are marked
###Poreins in this list have been reported in Nagaraja et al's experiment.
The secrometic experiment of the present invention indentified some of the well-characterized breast cancer related cytosolic proteins such as Cyclophilin A, 14-3-3 delta and peroxiredoxin 2 in culture media. It wa essential to validate the levels of these cytosolic proteins in the medium from independent experiments. To this end, the expression level of cyclophilin A, 14-3-3 delta and peroxiredoxin 2 from the culture media of MDA-MB-231, MCF-7 and MCF-10A were validated with immunoblotting. The results indicated that both the proteomic and immunoblot analysis showed cyclophilin A and 14-3-3 delta down-regulated in MCF-7 in comparison to the levels in MCF-10A. In contrast, peroxiredoxin 2 showed up-regulation in MCF-7 in comparison to the levels in MCF-10A. Comparing the secreted protein levels between MCF-10A and MDA-MB-231 indicates that the peroxiredoxin 2 and 14-3-3 delta expression levels increased in MDA-MB-231 and MCF-10A, respectively; however, the cyclophilin A level showed no significant change (
Immunoblot and immunofluorescence analyses were carried out to further confirm the differential protein levels observed in the total cellular proteins (profilin, cathepsin D, annexin 2, protein disulfide isomerase A1 and HDAC1) across MDA-MB-231, MCF-7 and MCF-10A (
The cellular proteomic and secretomic analyses above reveal a number of identified proteins may be breast cancer markers (Tables 1 and 2). To verify this observation, immunoblotting and immunofluorescence were used to validate these differentially expressed proteins including bestrophin 3, MPP2, parvalbumin, PdLIM1, IFIT3 and BANF1 as these proteins showed relatively significant changes (>3 fold) in comparison with most of the unreported identified proteins across MCF-10A, MCF-7 and MDA-MB-231. The immunoblotting analysis of concentrated serum-free media shows that more bestrophin 3 was secreted in the cell lines of MCF-7 and MDA-MB-231 than MCF-10A, while MPP2 was only detected in MDA-MB-231. Notably, the bestrophin 3 blotting result did not completely agree with the 2D-DIGE data, where levels in MCF-7 were higher than MB-231 (
With the basis of a Swiss-Prot search and KEGG pathway analysis, numerous potential biological functions of the identified proteins across MCF-10A, MCF-7 and MDA-MB-231 were determined. The information should be useful for studying the mechanisms of breast cancer tumorigenesis and metastasis.
Other differentially expressed proteins of interest across MCF-10A, MCF-7 and MDA-MB-231 include cathepsin D, bestrophin-3 and interferon-induced protein with tetratricopeptide repeats 3 (IFIT3). Cathepsin D, a lysosomal aspartic protease, is over-expressed in estrogen receptor positive breast cancer cells and is generally of good prognostic value in comparison with estrogen receptor negative breast cancer in clinical studies. The present invention indicates that cathepsin D is highly expressed in MCF-7, both in total cellular proteins or in secreted fraction. In contrast, cathepsin D is significantly down-regulated in MDA-MB-231 cells compared with MCF-7.
Results of the proteomic experiments display good correlation with secrmetic experiments.
Bestrophin-3, a cGMP-dependent calcium-activated chloride channel, has not been reported to be associated with cancer and shows upregulation in MCF-7 and MDA-MB-231 in the present inveniton. Nevertheless, the related study in bestrophin-1 showed the protein improves intracellular Ca2+ signaling and increases cell growth rate in colonic carcinoma cells. The proliferation of the cells was significantly suppressed by bestrophin-1 RNA interference treatment (Spitzner, M.; Martins, J. R.; Soria, R. B.; Ousingsawat, J.; Scheidt, K.; Schreiber, R.; Kunzelmann, K. J. Biol. Chem. 2008, 283, 7421). This indicated that bestrophin-3 may be a potential target for breast cancer therapy. IFIT3 plays a key role in the antiproliferative activity of the interferon-related signaling pathway through inducing expression of cell cycle inhibitors, p21 and p27 proteins (Xiao, S.; Li, D.; Zhu, H. Q.; Song, M. G; Pan, X. R.; Jia, P. M.; Peng, L. L.; Dou, A. X.; Chen, G. Q.; Chen, S. J.; Chen, Z.; Tong, J. H. Proc. Natl. Acad. Sci. U.S.A 2006, 103, 16448). The 2D-DIGE results in this invention showed that IFIT3 is downregulated in both MCF-7 and MDA-MB-231 cells, implying that breast cancer cells may maintain a high level of proliferative activity by downregulating the expression of IFIT3.
Based upon the data presented here, the present invention infers that proteins selected from biomarker library plays key role in the regulation of breast cancer progression. Combining the data of 2D-DIGE and immunoassay, it concludes that the increasing or decreasing of biomarker expression relative to the expression of normal tissue results in an increased likelihood of breast cancer development.
