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Tumor Biology |
Women’s Cancers Section, Laboratory of Pathology, National Cancer Institute, Bethesda, Maryland 20892 [J. D. W., K. McL., K. McG., M. K., P. S. S.]; Department of Pathology, Harvard Medical School, and the Molecular Pathology Unit, Massachusetts General Hospital, Boston, Massachusetts 02129 [D. C. S.]; Laboratory of Pathology, National Cancer Institute, Bethesda, Maryland 20892 [H. K., M. J. M.]; Department of Biochemistry, U. T. Southwestern Medical Center, Dallas, Texas 75390 [S. C., H. S., Y. Z.]; Department of Pathology, U. T. M. D. Anderson Cancer Center, Houston, Texas 77030 [A. S.]; Department of Obstetrics and Gynecology, University of Tuebingen, Tuebingen, D-72076 Germany [R. K., D. W.]; and Food and Drug Administration-National Cancer Institute Clinical Proteomics Program, Division of Therapeutic Proteins, Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, Maryland 20892 [E. F. P.]
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ABSTRACT |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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A number of hormone/growth factor receptor, angiogenesis, apoptosis, oncogene, or suppressor proteins germane to infiltrating ductal breast cancer have been investigated by immunohistochemical or similar methods in formalin-fixed, paraffin-embedded sections of DCIS, some of which also included a comparison to normal ductal/lobular units (16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30) . High throughput technologies such as arrays, SAGE, and proteomics offer the potential to find alterations previously unidentified in the breast or in other cancers. Technical challenges abound, including the requirement for promptly frozen material and lesion size. Two nucleic acid-based analyses of DCIS have been reported and included four matched cases of normal ductal/lobular units (31 , 32) .
We report the first proteomic analysis of matched normal ductal/lobular units and DCIS. Proteomic evaluations in their simplest form use two-dimensional gel electrophoresis to identify differentially expressed protein spots, which are sequenced by MS. Variations can also detect posttranslational modifications of proteins such as phosphorylation or ubiquitination (reviewed in Refs. 33, 34, 35 ). This technique offers the advantages of detection at the functional level of protein expression and the ability to interrogate subcellular distribution, protein complex formation, and pathways such as signal transduction and degradation. Disadvantages of the technique include its labor intensive nature and the dynamic range of biological protein mixtures in which the most abundant proteins are preferentially identified on gels. To improve the detection of low abundance proteins, lysates of both whole tissue sections and up to 100,000 LCM epithelial cells were separated on two-dimensional gels. We report herein a list of differentially expressed proteins and validation efforts. Our data identify novel protein expression or modification trends in DCIS, which are distinct from the results of nucleic acid-based approaches, and which paint a new portrait of this disease.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Tissue Processing and LCM.
Frozen blocks of patient-matched breast tissue containing normal ductal epithelium and DCIS lesions were cut into 8.0-µm sections, placed on uncoated glass slides, and stored at -80°C. Immediately before dissection, sections were fixed in 70% ethanol for 8 s, stained with hematoxylin for 8 s, dehydrated for 10 s each in 70, 95, and 100% ethanol followed by xylene for a minimum of 2 min. All solutions for staining, except xylene, were supplemented with protease inhibitor tablets (Roche Molecular Biochemicals, Indianapolis, IN). The sections were air-dried and microdissected with a PixCell I or II Laser Capture Microdissection System (Arcturus Engineering, Mountain View, CA). Approximately 50,000 or 100,000 cells each of normal breast epithelium and DCIS cells were microdissected and stored on microdissection caps at -80°C until lysed for two-dimensional gel electrophoresis. Each cell population was determined to be >95% homogeneous by microscopic visualization of the captured cells.
Two-Dimensional Gel Electrophoresis.
Tissue proteins were separated by IEF over a pH 3–10 range followed by gradient SDS-PAGE. Microdissected cell populations or corresponding whole tissue sections (8-µm sections, 10–15/sample) were lysed directly into buffer containing 7.0 M urea, 2.0 M thiourea, 4% (w/v) 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid, 1% (w/v) decanoyl-N-methyl-glucamide (MEGA-10), 1% (w/v) octyl-b-glucopyranoside, 0.5% (v/v) Triton X-100, 50 mM DTT, 1% (v/v) pH 3–10 NL IPG Buffer (Amersham-Pharmacia Biotech, Piscataway, NJ), 1% (v/v) ß-mercaptoethanol, 2.0 mM tributylphosphine, and 40 mM Tris-HCl. For large format IEF, samples were lysed in a total volume of 400 µl and loaded onto 18-cm, pH 3–10 NL IPG strips, and for small format IEF, 150 µl of lysis buffer and 7-cm, pH 3–10 NL IPG strips were used. IEF was conducted using a Multiphor II system (Amersham-Pharmacia Biotech) according to manufacturer’s instructions. Focused strips were equilibrated with 6.0 M urea, 26 mM DTT, 4% SDS, 30% glycerol in 0.1 M Tris-HCl (pH 6.8) for 15 min followed by 6.0 M urea, 0.38 M iodoacteamide, 4% SDS, 30% glycerol, and a dash of bromphenol blue in 0.1 M Tris-HCl (pH 6.8) for 10 min. The equilibrated large format strips were applied directly to 9–18% gradient SDS-polyacrylamide gels and separated overnight at 40V constant voltage. Small format strips were applied to 4–20% gradient Zoom SDS minigels (Novex/Invitrogen, Carlsbad, CA) and electrophoresed at 80–100V. Gels of microdissected samples were fixed and stained with GelCode Blue Coomassie stain (Pierce, Rockford, IL) overnight and destained in H2O followed by silver staining (36)
. Gels of whole tissue sections were fixed in 7% methanol, 10% acetic acid for 30 min, and stained with Sypro Red (Molecular Probes, Eugene, OR) overnight. After rinsing in three changes of H2O for 1 h, the gels were scanned with a Fluorimager SI (Molecular Dynamics, Sunnyvale, CA). The gels were then rinsed extensively in H2O and stained with GelCode Blue as described.
Two-Dimensional Gel Analysis and MS Sequencing.
All Coomassie and silver-stained gels were scanned into Adobe Photoshop 6.0 (Adobe, San Jose, CA) with a Umax PowerLookIII scanner (Umax, Dallas, TX) and printed. Two independent reviewers analyzed prints of the gels by visual inspection for protein expression differences between microdissected normal and DCIS cells as well as whole normal and tumor tissue sections. Differences in protein levels were defined as clear visual differences in size and/or density of the protein spot on the gel. Protein spots of interest from microdissected gels were identified on gels of whole tissue sections and excised. In-gel proteolytic digestion and MS sequencing of protein spots were performed as described previously (37)
. The Phoretix Two-Dimensional Advanced v5.01 analysis program (Nonlinear Dynamics, Durham, NC) was used to estimate the relative differences in spot intensity for candidate proteins. Cropped regions of normal and DCIS gels containing each candidate protein were made. Protein spots in the cropped images were detected, spots from normal and DCIS gels matched, and individual spot volume values were obtained according to the program instructions. Three or more additional sets of matched spots were also identified in each set of cropped images and their volumes set equal to 1.0 using the program’s volume normalization function. Ratios of the normal versus DCIS normalized volume values for the candidate proteins were compared with each other, and a mean relative difference in spot intensity was calculated.
Validation of Candidate Proteins.
Amino acid sequences corresponding to tryptic peptide masses identified in candidate proteins were subjected to BLAST homology searches to rule out alternative protein identifications. Antibodies against confirmed candidate proteins were obtained and screened for specificity by immunoblot analysis of human breast culture cell lysates and whole breast tumor tissue lysates. Approximately 50 µg of protein were separated on SDS gels and transferred to polyvinylidene difluoride membrane. Blots were blocked overnight with 5% nonfat dry milk in PBS (pH 7.4). Primary antibodies were diluted according to the manufacturer’s or provider’s recommendations into 5% nonfat dry milk, 0.1% Tween 20 in PBS, and incubated with blots at room temperature for 3 h. Blots were washed 3 x 5 min in 0.1% Tween 20 in PBS. Horseradish peroxidase-conjugated secondary antibodies (Zymed Laboratories, South San Francisco, CA) were diluted 1:7500–10,000 in milk/Tween 20/PBS and incubated at room temperature for 1 h. Blots were washed 3 x 5 min in 0.1% Tween 20/PBS, followed by development with enhanced chemiluminescence reagents (Amersham-Pharmacia Biotech) and exposure to Biomax ML film (Eastman Kodak, Rochester, NY).
IHC.
Antibodies that recognized a single band of the appropriate molecular weight on immunoblots were used for IHC. Five-µm sections of frozen-infiltrating ductal carcinomas containing both normal and DCIS tissue were cut and placed on charged microscope slides. Tissue was thawed and fixed in 4% paraformaldehyde in PBS for 10 min followed by a 10-min incubation in PBS supplemented with 300 mM sucrose, 20 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, 50 mM NaCl, 3 mM MgCl2, and 0.5% Triton X-100 (38)
. Slides were rinsed 3 x 5 min in PBS and then treated with 0.3% H2O2 in PBS for 30 min for monoclonal antibody staining or 1.0% H2O2 in PBS for 20 min for polyclonal antibody staining. Tissue was rinsed 5 min in PBS and blocked with 10% serum in PBS for 30 min. Sections were incubated with primary antibodies diluted in 10% serum in PBS as follows: Annexin II (no. 0590–5036; Biogenesis, Inc., Brentwood, NH), 1:20 overnight; Annexin V (no. 0590–5109; Biogenesis, Inc.), 1:700 for 3 h; Annexin VII (no. 610668; BD Transduction Labs, San Jose, CA), 1:25 for 3 h; CLIC-1 (Ref. 39
; gift of Dr. John Edwards from St. Louis University, St. Louis, MI), 1:50 for 3 h; GRP78 (no. sc-1050; Santa Cruz Biotechnology, Inc., Santa Cruz, CA), 1:20 for 3 h; Hsp 27 (no. ms-101-p0; Neomarkers, Inc., Freemont, CA), 2 µg/ml for 3 h; Hsp 90 (no. sc-7947; Santa Cruz Biotechnology, Inc.), 1:100 for 3 h; Peroxiredoxins I and II (Ref. 40
; gift of Dr. Sue Goo Rhee from NIH, Bethesda, MD), 1:400 and 1:1000, respectively, 3 h; Profilin (no. 210-740-r100; Alexis Biochemicals, San Diego, CA), 4 µg/ml for 3 h; Prohibitin (no. rb-292-p0; Neomarkers, Inc.), 50 µg/ml for 3 h; Rab 11 (no. 610656; BD Transduction Labs), 1:20 overnight; Stathmin (no. 611146; BD Transduction Labs), 1:10 overnight; Transgelin (Ref. 41
; gift from Dr. Michael Parmacek from University of Pennsylvania, Philadelphia, PA) 1:1000 for 3 h; and VDAC/Porin 31HL (no. 529532; Calbiochem, San Diego, CA), 1:25 for 3 h. Slides were rinsed in PBS and the Vectastain avidin-biotin complex kit and 3,3'-diaminobenzidine kit (Vector Laboratories, Burlingame, CA) were used according to manufacturer’s instructions to visualize antibody localization. Negative controls for all cases were incubated only with the labeled secondary antibody provided in the avidin-biotin complex kit. Tissue sections were counterstained with hematoxylin for 5 min, dehydrated with 100% ethanol and xylene, and coverslips mounted with Permount. IHC staining was evaluated by a pathologist and a molecular biologist for staining intensity, and normal, DCIS, and invasive tissue were scored on a 0–3+ scale.
Comparison to SAGE Analysis.
An online SAGE analysis was conducted at the Web site.5
Four sources of normal breast cells (SAGE mammary epithelium, SAGE Duke 40N, SAGE Duke 48N, and SAGE Br N) containing 70,118 tags were queried with two sources of DCIS (SAGE DCIS and SAGE DCIS2), containing 105,958 tags. The 100 SAGE tags most likely to be at least 2-fold different were listed using the Web site’s directions.
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RESULTS |
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. Four cases were pure DCIS, and two were DCIS associated with infiltrating ductal carcinoma. Although most of the cases were large to obtain sufficient material, several cases were smaller (1–2 cm); one lesion was a less advanced stage II grade. The patients were relatively young. Breast cancers from young women have been reported to be histopathologically and clinically distinct (42, 43, 44)
. The normal ductal/lobular units from two cases were exceptional. In case 4, the normal ductal cells exhibited gestational hyperplasia. This unique case allowed us to examine protein expression differences between two matched proliferative cell types, one in a regulated differentiation process and one cancer. Another interesting case (case 2) represented a double mastectomy for DCIS. The DCIS protein profile was compared with those of ipsilateral and contralateral ductal/lobular cells.
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Limited tissue availability and the large quantities of material required for microdissection precluded running duplicate gels of whole cryostat sections or microdissected tissue of the cases obtained for this study. However, the availability of two sources of normal ductal/lobular tissue from a single patient (Table 1
, case 2) allowed us to assess the spot patterns from samples that we predict should be highly similar. Indeed, the two-dimensional gels from whole cryostat sections of the ipsilateral and contralateral normal specimens showed that 89% of 250 of the visible spots were present in both gels. Analysis of 200 of the visible spots from two-dimensional gels of the microdissected normal specimens showed 91.3% were similar. These analyses validated our methods for sample processing for two-dimensional gels and confirmed our ability to obtain highly similar, if not identical, spot patterns on two-dimensional gels from related samples.
The accuracy of protein spot collection for sequencing was also assessed. For a subset of the differentially expressed proteins (n = 32), the corresponding spots were excised from both the normal and DCIS gels for sequencing; 30 of 32 sets (94%) yielded identical protein sequences, confirming the accuracy of sample collection and sequencing.
Proteomic Analysis.
Ten sets of two-dimensional gels were compared, based on two different strategies to maximize the identification of lower abundance proteins. For all six cases, multiple frozen sections containing either normal or DCIS tissue were lysed for two-dimensional gel analysis. This strategy permitted a large amount of total protein to be loaded on gels to optimize detection and sequencing. These gels were also used to identify potential differences in nonepithelial compartments such as the extracellular matrix. In four cases (cases 2, 4–6), sufficient normal and DCIS tissue was available for LCM-based analysis of normal and DCIS epithelial cells. These gels, which contained up to 100,000 laser-captured cells (up to 40,000 laser pulses/sample), were silver stained to increase the sensitivity of spot detection and served as a map for the excision of differentially expressed proteins from the corresponding whole tissue section gels for MS analysis. Two-dimensional gels of DCIS from case 4 demonstrated that the microdissected epithelial cells produced a distinct protein profile from those of whole tissue sections (Fig. 1)
. This general observation was borne out in our MS sequencing: only four proteins were identified with the same pattern of differential expression in gels from both LCM material and whole tissue sections (GRP78, Hsp 27, hnRNP-L, and 3-hydroxyisobutyrate dehydrogenase), indicating that the use of two comparison strategies increased the diversity of useful identifications.
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. Fig. 2
shows cropped images from two-dimensional gels demonstrating the differential expression of selected proteins. The MS peptide(s) obtained for each protein spot were queried in BLAST searches to ensure that each identification corresponded to a single protein. The 55 known proteins are grouped by function; two MS sequences corresponded to theoretical proteins in BLAST searches. Table 2
also lists a densitometric estimate of relative intensity differences, which ranged from modest (2-fold) to >400-fold. The Phoretix Two-Dimensional Advanced v5.01 image analysis program normalized the size and intensity difference of the candidate protein spot on the two gels to that of multiple additional matched protein spots in the geographic vicinity of the differentially expressed spot.
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Confirmatory Studies.
Three approaches based on protein expression levels were used to validate the differential expression trends observed in the two-dimensional gels. First, 10 of 57 differentially expressed proteins were observed in two-dimensional gels from two or more independent cases (Table 2)
. Second, literature searches confirmed the differential expression patterns of Hsp 27 (45)
, GRP78 (46)
, and cathepsin B (47)
between normal ductal/lobular units and DCIS.
Third, IHC was used to validate the expression patterns of 15 proteins. Limited data exists in the literature to indicate whether differentially expressed protein spots on two-dimensional gels, particularly in the lower ranges of altered expression, translate into observable intensity differences on traditional IHC. IHC was conducted on frozen infiltrating ductal breast carcinomas because (a) limited amounts of published IHC data existed for many of the antibodies used and (b) the enhanced probability of artifacts resulting from antigen retrieval used in formalin-fixed, paraffin-embedded specimens. An independent cohort of infiltrating ductal breast carcinomas was used. H&E-stained sections of 75 carcinomas were examined by a pathologist, and six were found to also contain normal ductal/lobular units and DCIS. The specificity of each antibody was verified on immunoblots using lysates of tissue culture cell lines and human breast tumors. Validation of IHC included antibody dilution experiments and control antibodies. The immunohistochemical staining of each antibody on the cohort was evaluated by a molecular biologist and a pathologist, and the staining intensity in the matched normal ductal/lobular units, DCIS and infiltrating components were determined on a traditional 0–3+ scale. Table 3
lists the comparisons of normal ductal/lobular unit and DCIS protein expression for these antibodies, and Fig. 3
presents selected photomicrographs. Of the 15 proteins examined, 14 of 15 confirmed the two-dimensional gel proteomic trend in at least one of the cases, and 12 of 15 confirmed the trend in 3 or more of the cases examined (Table 3
, Fig. 3
). IHC confirmation of two-dimensional proteomic trends was observed for both the DCIS>N (Fig. 3, A, B, E, and F)
and reverse (Fig. 3, C and D)
patterns. Proteins exhibiting limited overexpression on two-dimensional gels such as annexin VII (4-fold) were also confirmed by IHC (Fig. 3, E and F)
. In all cases, protein expression in the infiltrating ductal component was comparable with that of DCIS (data not shown). The only protein expression trend not confirmed was VDAC, which exhibited multiple spots on the two-dimensional gels and may reflect posttranslational processing. Of the 55 known proteins listed in Table 2
, 21 of 55 have been confirmed.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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To date, the majority of the DCIS literature has followed that of infiltrating ductal (invasive) breast carcinoma, where growth factor and hormone receptors, angiogenesis and p53 mutations have been studied. We report the first proteomic analysis of matched normal breast cells and DCIS and identify a new list of targets, many previously unconnected with invasive disease. Proteomic and other high throughput molecular analyses are hypothesis-generating experiments. The sorting of our differentially expressed proteins into several functional groups (Table 2)
permitted the development of hypotheses concerning their potential involvement in breast oncogenesis.
We report numerous and heterogeneous alterations in actin-binding protein expression in DCIS. This finding was considered unusual because the actin cytoskeleton is typically studied in lamellar protrusions in motility and invasion, and DCIS is defined as a preinvasive lesion. DCIS lesions exhibited increased expression of barbed-end capping proteins, the Arp3 component of the actin nucleation complex, the depolymerization protein cofilin, the actin-bundling protein L-plastin, and profilin, which is thought to present actin-ATP monomers to the extending barbed end; expression of the cross-linking protein transgelin was decreased. IHC studies confirmed the transgelin and profilin two-dimensional patterns. Several aspects of this list are noteworthy: the minimal cellular requirements for actin filament assembly involved in Listeria motility included actin monomer, Arp2/3 complex, cofilin, and capping protein (52)
. The majority of these components are represented in Table 2
, suggesting that functional differences have been observed. The proteins CapG, CapZ, profilin, and cofilin bind and are functionally modulated by the lipid signaling intermediate PIP2, tying the potential actin cytoskeletal alterations in DCIS to signal transduction. Transfection studies have demonstrated the functional effects of increased expression of single actin regulatory proteins in various cell types (53, 54, 55)
, although the overexpression of a combination of actin regulatory proteins has not been reported. Preliminary immunofluorescence data using frozen sections of breast tissue suggest increased F-actin localization at the membrane in DCIS, as compared with normal ductal/lobular units.6
Because DCIS is defined by a lack of histological evidence of invasion, the data suggest that functions other than the typical lamellar protrusions in invasion and motility may be impacted. The Arp 2/3 complex has been reported to nucleate actin for vesicle transport in response to Rho family proteins (56)
, suggesting that alterations in the DCIS cytoskeleton may relate to vesicular trafficking. Robust (136-fold) overexpression of stathmin, a microtubule destabilizing protein, was found in the two-dimensional gel analysis and confirmed by IHC, suggesting that the microtubule network may also be altered in DCIS.
Our data propose that DCIS is a disease of deregulated lipid, vesicular, and membrane trafficking. Rab 11a and three annexins were increased in DCIS 3–21-fold relative to matched normal ductal/lobular cells, and all were confirmed by IHC. Rab 11a is a GTPase involved in recycling ligand receptors back to the cell surface, transport through the trans-Golgi network, and some aspects of exocytosis. It is of interest that a product of Rab 11a vesicular trafficking in breast cells, cathepsin B (57) , was coordinately up-regulated in case 3 DCIS. Transfection studies demonstrate that elevated Rab 11 expression results in altered localization of membrane proteins and exocytosis (58 , 59) . The annexins identified are calcium- and phospholipid-binding proteins that function in lipid rafts and in secretory vesicle fusion. Autocrine production of, and responsiveness to growth factors has been hypothesized to contribute to breast cancer (60) . The roles of growth factor secretion, receptor presentation in membranes, and receptor recycling will be of interest in this context.
The hypothesis of DCIS as a disease of altered intracellular trafficking also extends to proteins, ions, fatty acids, and retinoids (Table 2)
. Of note are proteins that bind and channel two potential cancer prevention compounds, retinoids (CRABP2) and selenium (selenium binding protein), suggesting that their bioavailability or signaling pathways in DCIS may be disrupted. The immunoglobulin receptor, with lower expression in DCIS, is involved in the transport of IgA across the breast epithelium into the duct via highly organized basolateral-to-apical vesicle trafficking (61)
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Two- to 26-fold overexpression of six different chaperone proteins was observed in DCIS, and four were confirmed by IHC. Our data extend published trends to chaperones in multiple intracellular compartments, including the endoplasmic reticulum and the mitochondria. The GRPs are involved in binding peptides and are thought to influence immunogenicity. Transfection data demonstrate roles for Hsp 90 in steroid receptor function (62 , 63) and for Hsp 27 in apoptosis.
The cellular microenvironment is widely reported to regulate mammary growth and differentiation, and it is altered by proteases during invasion. Little is known of the breast extracellular microenvironment preinvasion. We were surprised by the decreased expression of the 
1 and 2 chains of type VI collagen in DCIS. The type VI collagen 3 chain was underexpressed in case 3 DCIS but was likely a breakdown product because of its molecular size and eliminated from further consideration (data not shown). Type VI collagen is distinguished from other more commonly studied forms of collagen by its nonfibrillar, supercoiled structure. It is commonly found around mesenchymal cells and is involved in the formation of a vascular network in melanoma (64)
. The only report of type VI collagen expression found in the breast literature noted its increased expression in the extracellular matrix remodeling in gynecomastia (65)
. Limited changes in protease expression were found in DCIS, including the overexpression of cathepsin B, the reduced expression of protease inhibitors, and an increase in mechanisms for the presentation of proteases at the tumor cell surface.
The influence of apoptosis on breast cancer development is suggested by several proteomic trends. Three peroxiredoxins were identified in our analysis, which reduce reactive oxygen species. Peroxiredoxins 1 and 2 were 454- and 8-fold overexpressed in DCIS, respectively, and were confirmed by IHC. Increased expression of both forms has been demonstrated to protect cells from apoptosis induced by hydrogen peroxide (66 , 67) . Our data suggest that posttranslational alterations occur in VDAC. VDAC is a receptor for the Bax and Bak proapoptotic factors and commits to apoptosis via outer mitochondrial membrane permeabilization; it is unstudied to date in breast cancer.
Some of the remaining proteins overexpressed in DCIS are well-known signal transduction proteins, including protein phosphatase 2A, 14-3-3 and RhoGDI. We find the overexpression of multiple hnRNPs of interest as they contribute to functions such as mRNA processing and telomere maintenance that have a role in genomic instability. The hnRNPs have been reported as markers of early lung cancer (68) . Of the S100 proteins identified, S100A11 exhibits a calcium-independent phosphorylation and nuclear translocation (69) , which may signify a distinct function.
The application of proteomic technologies to clinical problems is relatively new. The approach of two-dimensional gel analysis of tissue specimens and MS sequencing remains inefficient because only 57 proteins of interest emerged from >300 potential spots. However, using our stringent criteria for inclusion, 10 protein spots were observed in >1 DCIS case and 14 of 15 trends from two-dimensional gels were validated immunohistochemically. The cohort size for both the proteomic analysis and IHC validation was small and statistical analysis would be uninformative. Our data also provide a first indication, other than anecdotal data using a single antibody, that proteomic trends from two-dimensional gels can be validated using techniques common in clinical practice such as IHC. With further study to validate antibodies on formalin-fixed, paraffin-embedded sections, these findings will enable wider study of proteomic hits to include larger cohorts of DCIS specimens, cohorts of premalignant lesions, and DCIS specimens from clinical trials linked to recurrence data.
Our data indicate that proteomic analyses provide unique information compared with nucleic acid-based technologies. Luzzi et al. (31) reported microarray analysis of three sets of matched normal ductal/lobular units and DCIS using LCM and amplification techniques. The only overlapping identification with our data were lactoferrin. A SAGE analysis of breast cancer progression included two DCIS samples, one of which was accompanied by matched normal ductal/lobular units (32) . The list of differentially expressed genes generated contained no overlap to our proteomic identifications. An online SAGE analysis (see "Materials and Methods") produced confirmatory data for Hsp 27 and S100A7. We do not believe that the lack of concordance is solely because of the limited dynamic range of proteomics, given our use of LCM-enriched cell populations and identification of low expression level proteins such as RhoGDI and mitochondrial proteins. Given the current widespread use of nucleic acid-based technologies for cancer research, our data suggest caution in the assumption that mRNA levels reflect those of functional proteins.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 This work was supported by the Early Detection Research Network Grant CA 85146 of the National Cancer Institute (to Y. Z). 
2 Present address: University of Colorado School of Health Sciences, Denver, CO 80262.
3 To whom requests for reprints should be addressed, at Building 10, Room 2A33, National Cancer Institute, Bethesda, MD 20892. Phone: (301) 402-2732; Fax: (301) 402-8910; E-mail: steegp{at}mail.nih.gov
4 The abbreviations used are: DCIS, ductal carcinoma in situ; SAGE, serial analysis of gene expression; MS, mass spectrometry; LCM, laser capture microdissected; IEF, isoelectric focusing; NL IPG, Nonlinear immobilized pH gradient; IHC, immunohistochemistry; VDAC, voltage-dependent anion channel; ER, estrogen receptor; Arp, actin-related protein; CRABP2, cellular retinoic acid-binding protein 2; GRP, glucose-regulated protein; HnRNP, heterogeneous nuclear ribonucleoprotein; Hsp, heat shock protein; RhoGDI, Rho guanine nucleotide dissociation inhibitor.
5 Internet address: www.ncbi.nlm.nih.gov/SAGE/sagexpsetup.cgi.
6 P. S. Steeg and J. D. Wulfkuhle, personal communication.
Received 7/ 9/02. Accepted 9/20/02.
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M. Gronborg, T. Z. Kristiansen, A. Iwahori, R. Chang, R. Reddy, N. Sato, H. Molina, O. N. Jensen, R. H. Hruban, M. G. Goggins, et al. Biomarker Discovery from Pancreatic Cancer Secretome Using a Differential Proteomic Approach Mol. Cell. Proteomics, January 1, 2006; 5(1): 157 - 171. [Abstract] [Full Text] [PDF]
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 L. Espana, B. Martin, R. Aragues, C. Chiva, B. Oliva, D. Andreu, and A. Sierra Bcl-xL-Mediated Changes in Metabolic Pathways of Breast Cancer Cells: From Survival in the Blood Stream to Organ-Specific Metastasis Am. J. Pathol., October 1, 2005; 167(4): 1125 - 1137. [Abstract] [Full Text] [PDF]
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C. Gebhardt, U. Breitenbach, K. H. Richter, G. Furstenberger, C. Mauch, P. Angel, and J. Hess c-Fos-Dependent Induction of the Small Ras-Related GTPase Rab11a in Skin Carcinogenesis Am. J. Pathol., July 1, 2005; 167(1): 243 - 253. [Abstract] [Full Text] [PDF]
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 C J Fabian, B F Kimler, M S Mayo, and S A Khan Breast-tissue sampling for risk assessment and prevention Endocr. Relat. Cancer, June 1, 2005; 12(2): 185 - 213. [Abstract] [Full Text] [PDF]
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Y. P. Lim Mining the Tumor Phosphoproteome for Cancer Markers Clin. Cancer Res., May 1, 2005; 11(9): 3163 - 3169. [Abstract] [Full Text] [PDF]
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P. Carter, L. Smith, and M. Ryan Identification and validation of cell surface antigens for antibody targeting in oncology Endocr. Relat. Cancer, December 1, 2004; 11(4): 659 - 687. [Abstract] [Full Text] [PDF]
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H. Alexander, A. L. Stegner, C. Wagner-Mann, G. C. Du Bois, S. Alexander, and E. R. Sauter Proteomic Analysis to Identify Breast Cancer Biomarkers in Nipple Aspirate Fluid Clin. Cancer Res., November 15, 2004; 10(22): 7500 - 7510. [Abstract] [Full Text] [PDF]
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 E. Huber, D. Vlasny, S. Jeckel, F. Stubenrauch, and T. Iftner Gene Profiling of Cottontail Rabbit Papillomavirus-Induced Carcinomas Identifies Upregulated Genes Directly Involved in Stroma Invasion as Shown by Small Interfering RNA-Mediated Gene Silencing J. Virol., July 15, 2004; 78(14): 7478 - 7489. [Abstract] [Full Text] [PDF]
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 G. D. Leonard and S. M. Swain Ductal Carcinoma In Situ, Complexities and Challenges J Natl Cancer Inst, June 16, 2004; 96(12): 906 - 920. [Abstract] [Full Text] [PDF]
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 C. Torres-Cabala, A. Panizo-Santos, H. C. Krutzsch, H. Barazi, M. Namba, M. Sakaguchi, D. D. Roberts, and M. J. Merino Differential Expression of S100C in Thyroid Lesions International Journal of Surgical Pathology, April 1, 2004; 12(2): 107 - 115. [Abstract] [PDF]
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 H. J. Burstein, K. Polyak, J. S. Wong, S. C. Lester, and C. M. Kaelin Ductal Carcinoma in Situ of the Breast N. Engl. J. Med., April 1, 2004; 350(14): 1430 - 1441. [Full Text] [PDF]
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T. Tomonaga, K. Matsushita, S. Yamaguchi, M. Oh-Ishi, Y. Kodera, T. Maeda, H. Shimada, T. Ochiai, and F. Nomura Identification of Altered Protein Expression and Post-Translational Modifications in Primary Colorectal Cancer by Using Agarose Two-Dimensional Gel Electrophoresis Clin. Cancer Res., March 15, 2004; 10(6): 2007 - 2014. [Abstract] [Full Text] [PDF]
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M. Huber, I. Bahr, J. R. Kratzschmar, A. Becker, E.-C. Muller, P. Donner, H.-D. Pohlenz, M. R. Schneider, and A. Sommer Comparison of Proteomic and Genomic Analyses of the Human Breast Cancer Cell Line T47D and the Antiestrogen-resistant Derivative T47D-r Mol. Cell. Proteomics, January 1, 2004; 3(1): 43 - 55. [Abstract] [Full Text] [PDF]
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 Y.-P. Lim, L.-S. Diong, R. Qi, B. J. Druker, and R. J. Epstein Phosphoproteomic fingerprinting of epidermal growth factor signaling and anticancer drug action in human tumor cells Mol. Cancer Ther., December 1, 2003; 2(12): 1369 - 1377. [Abstract] [Full Text] [PDF]
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 J. Loscalzo Proteomics in Cardiovascular Biology and Medicine Circulation, July 29, 2003; 108(4): 380 - 383. [Full Text] [PDF]
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H. Hondermarck Breast Cancer: When Proteomics Challenges Biological Complexity Mol. Cell. Proteomics, May 1, 2003; 2(5): 281 - 291. [Abstract] [Full Text] [PDF]
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