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Cancer Research 66, 11194, December 1, 2006. doi: 10.1158/0008-5472.CAN-04-1444
© 2006 American Association for Cancer Research
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Cell, Tumor, and Stem Cell Biology

Identification and Validation of Differences in Protein Levels in Normal, Premalignant, and Malignant Lung Cells and Tissues Using High-Throughput Western Array and Immunohistochemistry

Jie Shen1, Carmen Behrens1, Ignacio I. Wistuba1,2, Lei Feng3, J. Jack Lee3, Waun Ki Hong1 and Reuben Lotan1

Departments of 1 Thoracic/Head and Neck Medical Oncology, 2 Pathology, and 3 Biostatistics and Applied Mathematics, The University of Texas M.D. Anderson Cancer Center, Houston, Texas

Requests for reprints: Reuben Lotan, Department of Thoracic/Head and Neck Medical Oncology-432, The University of Texas M.D. Anderson Cancer Center, Houston, TX 77030. Phone: 713-792-8467; Fax: 713-745-5656; E-mail: rlotan{at}mdanderson.org.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The identification of proteins, which exhibit different levels in normal, premalignant, and malignant lung cells, could improve early diagnosis and intervention. We compared the levels of proteins in normal human bronchial epithelial (NHBE) and tumorigenic HBE cells (1170-I) by high-throughput immunoblotting (PowerBlot Western Array) using 800 monoclonal antibodies. This analysis revealed that 87 proteins increased by >2-fold, and 45 proteins decreased by >2-fold, in 1170-I compared with NHBE cells. These proteins are involved in DNA synthesis and repair, cell cycle regulation, RNA transcription and degradation, translation, differentiation, angiogenesis, apoptosis, cell adhesion, cytoskeleton and cell motility, and the phosphatidylinositol 3-kinase signaling pathway. Conventional Western blotting using lysates of normal, immortalized, transformed, and tumorigenic HBEs and non–small cell lung cancer cell lines confirmed some of these changes. The expression of several of these proteins has been then analyzed by immunohistochemistry in tissue microarrays containing 323 samples, including normal bronchial epithelium, hyperplasia, squamous metaplasia, dysplasias, squamous cell carcinomas, atypical adenomatous hyperplasia, and adenocarcinomas from 144 patients. The results of the immunohistochemical studies correlated with the Western blotting findings and showed gradual increases (caspase-8, signal transducers and activators of transcription 5, and p70s6K) or decrease (E-cadherin) in levels with tumor progression. These results indicate that the changes in proteins detected in this study may occur early in lung carcinogenesis and persist in lung cancer. In addition, some of the proteins detected by this approach may be novel biomarkers for early detection of lung cancer and novel targets for chemoprevention or therapy. (Cancer Res 2006; 66(23): 11194-206)


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The leading cause of cancer death among both men and women, lung cancer is predicted to cause 163,510 deaths in the United States in 2005 (1, 2). The poor early detection of lung cancer coupled with ineffective treatments for advanced disease is responsible for the low (<17%) overall 5-year survival rate (1, 3). Therefore, new approaches to early diagnosis and prognosis of lung cancer are urgently needed (1, 3). The development of genomics has provided opportunities for global analysis of gene expression, and, indeed, several studies have identified differences in gene expression between normal and malignant lung cells using techniques such as serial analysis of gene expression (4) and cDNA microarray (5, 6). However, the levels of mRNA do not always reflect accurately the levels of the corresponding proteins nor do they reveal changes in epigenetic posttranscriptional modulation of proteins (e.g., phosphorylation, acetylation, methylation, ubiquitination, sumoylation, ADP ribosylation, glycosylation, myristoylation, isoprenylation, etc.) or changes in protein degradation rates. Therefore, analysis of differences in protein levels or modifications is important to complement studies on mRNA expression (7–9). Most proteomic studies have used two-dimensional PAGE combined with mass spectrometry (9, 10). More recently, surface-enhanced laser desorption ionization time-of-flight (TOF) mass spectrometry (MS) using chips with modified surface properties (11) has been used for detection of protein changes between different types of cancer (12–15). However, only limited studies have applied proteomic approaches to lung cancer (9, 16, 17). A recent study has used matrix-assisted laser desorption/ionization MS (MALDI-MS) directly from frozen tissue sections from resected non–small cell lung cancer (NSCLC) for profiling of protein expression to predict histologic groups as well as nodal involvement and survival (18). The specificity and high affinity of antibodies has been applied to proteomic analysis through an antibody-chip, which is a new approach to biomarker discovery for diagnosis and protein target identification (19). Another method known as BD PowerBlot Western Array Screening system (Becton Dickinson Biosciences, San Jose, CA) is a high-throughput Western immunoblotting method, which uses mixtures of subsets of about 800 monoclonal antibodies to evaluate differences in levels of cellular signaling proteins between total cell extracts from different cells or tissues. We used this method to identify protein changes between normal, immortalized, transformed, and tumorigenic lung epithelial cell lines and NSCLC cell lines and examined their differential expression in resected human lung specimens, including normal, premalignant, and malignant tissues, using immunohistochemistry.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cells and culture conditions. Normal bronchial epithelial cells (NHBE) were purchased from Whittaker-Clonetics (San Diego, CA). NSCLC cell lines H460, H1792, SK-MES-1, Calu-1, and A549 were obtained from Dr. Adi Gazdar (University of Texas Southwestern, Dallas, TX). Immortalized BEAS-2B cells (20) were obtained from Dr. Curtis Harris (National Cancer Institute, Bethesda, MD). Immortalized 1799, transformed 1198, and tumorigenic 1170-I bronchial epithelial cells derived from BEAS-2B as described by Klein-Szanto et al. (21) were obtained from Dr. Jonathan Kurie (University of Texas M.D. Anderson Cancer Center, Houston, TX) and Dr. Klein Szanto (Fox Chase, Philadelphia, PA). NHBE cells, the BEAS-2B cells, and the 1799 cells were grown in keratinocyte serum-free medium (Life Technologies, Inc., Rockville, MD) supplemented with bovine pituitary extract (50 µg/mL) and epidermal growth factor (5 ng/mL). The 1198 cells and the 1170-I cells were grown in keratinocyte serum-free medium supplemented with bovine pituitary extract and with 3% fetal bovine serum (FBS). NSCLC cell lines were maintained in a 1:1 mixture of DMEM/Ham's F12 medium supplemented with 5% FBS. The cells were maintained in a humidified incubator in a 95% air/5% CO2 atmosphere at 37°C.

BD PowerBlot Western Array. Total cellular proteins were extracted from NHBE and 1170-I cells using a lysis buffer containing 10 nmol/L Tris (pH 7.4), 1.0 mmol/L sodium orthovanadate, 1.0% SDS. Three milliliters of boiling lysis solution were added per 15-cm plate to cell monolayer that had been washed with PBS. The lysate was heated and sonicated for 10 to 30 seconds. The protein concentration was determined with the Protein Assay kit (Bio-Rad, Hercules, CA). Protein extracts were frozen and sent on dry ice to BD Biosciences for analysis. The samples were analyzed by SDS-PAGE in 16 x 16 x 0.1 cm, 5% to 15% gradient polyacrylamide slab gels. Cell extract containing 400 µg of protein was loaded in one well across the entire width of the gel. The gel was run overnight. The proteins were then transferred electrophoretically to Immobilon-P nylon membrane (Millipore, Bedford, MA) for 1 hour at 1 A. After transfer, the membrane was blocked for 1 hour with 5% milk. Next, the membrane was clamped with a Western blotting manifold that isolates 45 channels across the membrane parallel to the direction of electrophoresis. In each channel, a mixture of four to five antibodies was added and allowed to hybridize for 1 hour. Different sets of antibodies (templates A-D) were used for each membrane. The blot was washed and hybridized for 30 minutes with secondary goat anti-mouse horseradish peroxidase. The membrane was washed and developed with chemiluminescence using SuperSignal West Pico from Pierce Biotechnology (Rockford, IL). Chemiluminescence signals are captured using the Kodak Image Station CCD Camera. Differences in protein level are estimated by densitometric scanning and normalized using internal standards. The method is highly specific and can detect subnanogram levels of protein.

Protein extraction and Western blotting analysis. Cells were washed in PBS and lysed in a buffer containing 50 mmol/L Tris-HCl (pH 8), 150 mmol/L NaCl, 0.1% SDS, 1% NP40, 1 mmol/L phenylmethylsulfonyl fluoride, 5 µg/mL aprotinin, and 5 µg/mL leupeptin. After incubation on ice for 15 minutes and centrifugation at 12,000 rpm for 10 minutes, the supernatants were collected, and the protein concentration was determined with the Protein Assay kit (Bio-Rad). Protein (50 µg) was electrophoresed through a 10% polyacrylamide gel under nonreducing conditions and transferred to a nitrocellulose membrane (Bio-Rad) by electroblotting. After transfer, the blots were incubated with first antibodies at room temperature for 1 hour. Monoclonal antibodies to Akt, Annexin VI, basic fibroblast growth factor (bFGF), E-cadherin, CaMK kinase (CaMKK), caspase-8, caspase-9, cyclin-dependent kinase 1 (CDK1), cyclin D3, DFF45, Eg5, poly(ADP-ribose) polymerase (PARP), PDK1, p53BP2, p70S6K, phosphatidylinositol 3-kinase (PI3K), and p110{alpha} were obtained from BD Transduction Laboratories (Lexington, KY). Monoclonal antibodies to Bad and PTEN were obtained from Cell Signaling Technology, Inc. (Beverly, MA). Monoclonal antibody to p21 was obtained from Exalpha Biologicals (Boston, MA). Monoclonal antibody to Bax and p53 and polyclonal antibody to Bid were obtained from Oncogene Research Products (Boston, MA). Monoclonal antibodies to Bcl-X/L, Bcl-2, caspase-3, caspase-6, and caspase-7 and polyclonal antibodies to cyclin B1 and FGFR-1 were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Monoclonal antibody to ß-actin was obtained from Sigma Chemical Co. (St. Louis, MO). Antibody to galectin-3 was from Vector Laboratories (Burlingame, CA). The membranes were then washed with PBS and incubated with secondary antibodies for 1 hour at room temperature. The membranes were then washed with PBS, and antibody binding was detected using the enhanced chemiluminescence system (Amersham Biosciences, Piscataway, NJ) and Hyperfilm MP (Amersham). The films were subjected to scanning densitometry to quantitate band intensity by using the NIH Image software.

Case selection and tissue microarray preparation. Formalin-fixed paraffin-embedded specimens from surgically resected primary NSCLC at pathology stages I to IV from patients who had no prior chemotherapy or radiotherapy were obtained from the Lung Cancer Specialized Program of Research Excellence Tissue Bank at the M.D. Anderson Cancer Center. H&E-stained slides of these cases were reviewed by an experienced pathologist (I.W.) and selected to contain normal bronchial epithelium and premalignant lesions besides the cancers. The 323 tissue specimens collected from 144 patients included 29 adenocarcinomas and 30 squamous cell carcinomas (SCC). In addition, we selected 30 normal bronchial epithelia, 74 bronchial hyperplasias, 22 squamous metaplasias, 63 squamous dysplasias and carcinomas in situ (16 mild and moderate dysplasias, grouped as low grade and 47 severe dysplasias and carcinomas in situ, grouped as high grade), and 75 atypical adenomatous hyperplasias (AAH). Tumors and lesions were classified according the latest WHO 1999 criteria. We considered normal bronchial epithelium, hyperplasia, squamous metaplasia, low-grade dysplasia, high-grade dysplasia, and SCC as representing different steps in the development of SCCs. AAH was considered a premalignant lesion for adenocarcinoma development. Tissue microarrays were prepared with a manual tissue arrayer (Advanced Tissue Arrayer ATA100, Chemicon International, Temecula, CA) using 1-mm-diameter cores in triplicate for tumors and 1.5-mm cores for normal epithelial and premalignant lesions. Some cases were represented in the tissue microarray by several cores. One H&E-stained section for each tissue microarray prepared was examined to determine the presence of the corresponding histology.

Immunohistochemical stainings. Four-micrometer-thick histology sections from tissue microarrays were cut, deparaffinized using standard xylene, and hydrated through graded alcohol into water. Immunohistochemical analysis was done using monoclonal antibodies against E-cadherin (G-10, Santa Cruz Biotechnology) and p70s6k (Thr389, Cell Signaling Technology) and polyclonal antibodies against caspase-8 (E-20, Santa Cruz Biotechnology) and signal transducers and activators of transcription 5 (STAT5; C-17, Santa Cruz Biotechnology). Antigen retrieval was done for all markers using steaming at 95°C for 30 minutes in 10 mmol/L citrate buffer (pH 6; DakoCytomation Target Retrieval, DAKO Corp., Carpinteria, CA). Endogenous peroxidase and nonspecific binding were blocked by using 3% hydrogen peroxide in TBST (DAKO; wash buffer 10x) and with TBST diluted 1:10 in FBS for 30 minutes, respectively. Then, the sections were incubated with primary antibodies (diluted in TBST-FBS solution) for 2 hours at room temperature. Depending on the origin of the primary antibody, DakoCytomation Envision+ Dual Link System or LSAB+ (DAKO) were used as secondary antibodies, following the corresponding manufacturer's instructions. Diaminobenzidine was used as a chromogen, and commercial hematoxylin was used for counterstaining. Corresponding negative controls were incubated in the absence primary antibody.

A trained pathologist (I.W.) examined immunohistochemical expression of the antigens as follows: E-cadherin in the cell membrane, caspase-8 and STAT5 in the cytoplasm, and p70s6k in the nuclei. Both the extent and intensity of immunopositivity were considered for the membrane and cytoplasmic immunostaining, with intensity ranging from 0 to 3 and the extent of staining from 0% to 100%. The final staining score (range = 0-300) was obtained by multiplying both scores. For nuclear immunostaining, the percentage of positive nuclei in 200 cells examined was calculated. Representative examples of stained tissues were photographed under the microscope.

Statistical analysis. Descriptive statistics were used to quantify the protein fold change in the cell line studies. Summary statistics of each biomarker at each level of histology diagnosis were provided. The distribution of the immunohistochemical expression of the each marker at the various histologies was shown in box plots. Repeated-measures ANOVA models were fitted to estimate effect of histology on the biomarker scores using SAS (22). All tests are two sided. Ps < 0.05 were considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Comparison of protein levels in NHBE and 1170-I cells by PowerBlot analysis. Chemiluminescence images of two PowerBlot Western immunoblots obtained with a subset of the 800 antibodies (Fig. 1A and B ) show proteins with increased levels (CaMKK, procaspase-7, N-cadherin, nexilin, RANBP1, and p70s6k) or decreased levels ({gamma}-catenin) in the tumorigenic 1170-I cells relative to NHBE cells. Additional examples of individual proteins and internal control proteins from NHBE and 1170-I cell lysates, which were found to change after analysis of different blots probed with different subsets of antibodies, are presented in Fig. 1C and D. Table 1 presents the summary of such results obtained for proteins showing mostly >2-fold change (increase or decrease) in tumorigenic versus normal cells. Most of the changes were between 2- and 5-fold. However, some changes were as high as 31-fold increase and 20-fold decrease. The proteins found to be increased have been grouped in Table 1A according to their functions, including DNA synthesis and DNA repair, transcription and RNA degradation, translation, cell cycle regulation, apoptosis, cytoskeleton structure and cell motility, cell adhesion, angiogenesis, and other functions. Similarly, the proteins found to be decreased were grouped in Table 1B according to function, including transcription and RNA degradation, translation, cell cycle regulation, apoptosis, cytoskeleton structure and cell motility, cell adhesion, angiogenesis, and other functions.


Figure 1
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  		Figure 1. PowerBlot Western array analysis of proteins from normal (NHBE) and tumorigenic (1170-I) lung epithelial cells. Total protein extracts from each of the two cell types were analyzed by SDS-PAGE (extract from one cell type applied across the top of the slab gel) and processed for high-throughput immunoblotting using about 200 antibodies (Becton Dickinson). A and B, mixtures containing four to five antibodies from template B of this collection were added in each of 45 lanes of a manifold placed on top of the nylon membrane onto which the proteins were blotted (see Materials and Methods). The numbers above and below the gel indicate the lane number. The numbers on the right indicate the migration of proteins with defined molecular weights (lane 45). Several proteins with different levels in normal and tumorigenic cells were encircled and identified by name and arrows on the right and below each blot. C and D, examples of PowerBlot Western Array results for individual proteins from different blots probed with different templates. C, segments (lanes 15-17) of two immunoblots probed with the same template of antibodies showing decreased level of E-cadherin in 1170-I cells and no change in another protein (clathrin H) that was selected as an internal control (i.c.). D, examples of changes in levels of different proteins sampled from different blots. Each differentially expressed protein is accompanied by another unrelated and unchanged protein from the same blot as an internal control. The negative of the original data for increased contrast.

 

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  		Table 1.
 
Confirmation of protein changes by conventional Western blotting. A subset of 25 proteins was selected from Table 2 for validation using conventional Western blotting (one antibody per blot) under conditions that were optimal for each antibody (Fig. 2A-D ). This method confirmed the PowerBlot results for 20 of 25 (80%) of these proteins. They included Cdk1, cyclin B1, cyclin D3, bFGF, PDK1, CaMKK, Akt, p70s6K, Bax, procaspase-7, procaspase-8, DFF45, PARP, Annexin VI, p53, 53BP3, C-cbl, Eg5, TFIIB, and E-cadherin. The PowerBlot data on changes in the levels of procaspase-3, procaspase-6, 80K-H, p38{delta}, and Bid between NHBE and 1170-I cells could not be confirmed. During the validation experiments, we also compared the levels of different proteins in normal HBE cells and the isogenic family of cell lines that includes immortalized (BEAS-2B and 1799), transformed (1198), and tumorigenic (1170-I) HBE cells as well as several cell lines established from human NSCLC tumors (e.g., H460, H1792, SK-MES-1, A549, and Calu-1). Examples of the Western blotting results of proteins involved in cell cycle regulation, apoptosis, and PI3K/Akt signaling pathway are presented in Fig. 2 (note the lanes marked NHBE and 1170-I enclosed within dashed boxes). The levels of most of the proteins analyzed by Western blotting (e.g., E-cadherin, cyclin B1, procaspase-3, procaspase-7, procaspase-8, PARP, DFF45, 53BP2, PDK1, PI3K, CaMKK, p70s6k, and cyclin D3) were similar in 1170-I and the NSCLC cell lines. Other proteins (e.g., Annexin VI, Eg5, CDK1, p53, and Bax) showed different levels in 1170-I cells and NSCLC cell lines. The levels of PDK1 and Akt were similar in 1170-I cells and in two of four NSCLC cell lines.


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  		Table 2. Demographic and clinical-pathologic data regarding the lung cancer and respiratory epithelial tissue specimens studied for immunohistochemical analyses

 

Figure 2
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  		Figure 2. Conventional Western blotting analysis of proteins from normal, immortalized, transformed, tumorigenic, and malignant lung epithelial cell lines. Total cell proteins were extracted from normal NHBE cells and the indicated cell lines and separated in different lanes of the same slab polyacrylamide gel, and then processed for conventional Western blotting using one primary antibody at a time. Five different slab gels were analyzed with one antibody at a time. The primary antibodies were as follows: (A) E-cadherin, cyclin B1, CDK1 (cdc2), Eg5, and Annexin VI; (B) p53, 53BP2, PDK1, and Bax; (C) PI3K, CaMKK, Akt, p70s6K, and cyclin D3; (D) procaspase-8, procaspase-7, procaspase-3, PARP, and DFF45. Each blot was stripped and reprobed with antibodies against ß-actin as an internal control for loading.

 
A graphic comparison of the levels of proteins in the in vitro lung carcinogenesis model obtained after scanning the Western blots and quantitative analysis (Fig. 3 ) has shown that many of the changes observed with 1170-I cells are also observed in immortalized cells except for E-cadherin, which decreased only in transformed and tumorigenic HBE cells and Annexin VI, which increased in one of the immortalized cells (1799) but not in BEAS-2B (Fig. 3). For most proteins, changes seem to occur gradually as cells progress through stages of increased malignancy (Fig. 3).


Figure 3
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  		Figure 3. Quantitative depiction of differences in protein levels among normal, immortalized, transformed, and tumorigenic cells based on scans of the Western blots in Fig. 2. A, proteins involved in cell cycle and cell adhesion. B, p53-related proteins. C, proteins related to the PI3K/Akt pathway. D, caspases and caspase-3 substrates.

 
Confirmation of protein changes by immunohistochemical analysis of normal, premalignant, and malignant lung tissues. To begin to assess the relevance of the data obtained using the in vitro cell carcinogenesis model for changes in proteins during lung carcinogenesis in vivo, we did immunohistochemical analysis of the expression of E-cadherin, caspase-8, p70s6K, and STAT5 selected as representatives of proteins involved in cell adhesion, apoptosis, and growth factor signaling, respectively, using tissue microarrays containing 323 samples, including normal, premalignant, and malignant (NSCLC) tissue samples from lungs resected from 144 lung cancer patients (Table 2). Examples of the immunohistochemistry results are presented in Fig. 4 . The level of E-cadherin, which is high in normal bronchial epithelium, decreased progressively in premalignant tissue and carcinoma. In contrast, the levels of caspase-8, STAT5, and nuclear p70s6K increased in the premalignant and malignant tissue samples. The semiquantitative immunohistochemistry results are presented in Fig. 5 . The repeated-measures ANOVA showed that the level of E-cadherin decreased significantly in high-grade dysplasia and SCC as well as in AAH and adenocarcinoma relative to normal epithelium (Fig. 5A). The level of caspase-8 showed a trend for increase in high-grade dysplasia and a significant increase in SCC and adenocarcinoma; however, it decreased significantly in AAH (Fig. 5B). The level of p70s6K increased significantly in hyperplasia, high-grade dysplasia, and adenocarcinoma but did not show statistically significant changes in the other histologies (Fig. 5C). Cytoplasmic STAT5 level increased significantly in high-grade dysplasia, SCC, AAH, and adenocarcinoma, whereas the nuclear STAT5 showed increases in high-grade dysplasia, AAH, and adenocarcinoma but not in SCC and the other histologies (Fig. 5D and E). Because normal bronchial epithelial cells may not be the precursor of AAH and adenocarcinoma, we also compared the levels of expression of the different proteins in adenocarcinoma and AAH and found that the difference in the levels of caspase-8 and nuclear p70s6K were statistically significant, whereas the differences in the level of E-cadherin and cytoplasmic and nuclear STAT5 were not (Fig. 5). There were no correlations between the changes in protein levels detected by immunohistochemistry and the gender, smoking status, or tumor-node-metastasis stage of the cancer patients from whom the tissues were obtained.


Figure 4
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  		Figure 4. Representative examples of photomicrographs showing the immunohistochemical expression of E-cadherin, caspase-8, STAT5, and p70s6K in the sequential pathogenesis of human NSCLCs of the squamous cell carcinoma and adenocarcinoma histologies.

 

Figure 5
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  		Figure 5. Box plots of immunohistochemical expression scores for E-cadherin (A), caspase-8 (B), p70s6K (C), cytoplasmic STAT5 (D), and nuclear STAT5 (E) in the sequential pathogenesis of lung cancer. Columns, mean; bars, SE. Numbers under histology represent number of specimens (N) examined in each category. Hyp, hyperplasia; SqM, squamous metaplasia; LG-D, low-grade dysplasia; HG-D, high-grade dysplasia; ADCA, lung adenocarcinoma. NS, nonsignificant (P > 0.05). *, P = 0.01 to 0.05; **, P = 0.001 to 0.01; ***, P < 0.001.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The identification of biomarkers of early lung cancer is expected to improve the long-term survival of lung cancer patients. The application of high-throughput technologies to identify genes and proteins that are expressed differentially in tumor cells and normal tissues seems to be promising (23). Gene expression profiling is considered a standard procedure for high-throughput analysis of normal and malignant tissues. However, the reported lack of correlation between mRNA and protein expression has highlighted the importance of conducting parallel proteomics studies to complement cDNA or oligonucleotide array data. In this study, we compared and contrasted the levels of nearly 800 proteins related to cell growth, differentiation, and apoptosis in normal and tumorigenic human lung epithelial cells by using the PowerBlot technique, which has been used previously to identify protein changes in head and neck squamous carcinoma cells (HNSCC) after docetaxel (24) or retinoid (25) treatments. We detected changes in 135 proteins (87 increased and 48 decreased) in tumorigenic 1170-I cells relative to normal cells. These proteins execute diverse functions associated with DNA synthesis and repair, RNA transcription and degradation, translation, cell cycle regulation, and apoptosis, cytoskeleton structure, cell adhesion and motility, angiogenesis, and other functions. Understanding the mechanisms underlying the changes in the levels of these proteins and how they are related to lung carcinogenesis will obviously require additional experiments, such as functional analyses using gene silencing and gene transfection experiments.

Our analysis does not indicate whether a change in protein level is the result of increased gene expression or stabilization of mRNA or protein. Recently, Yoo et al. (24) found a 35% discrepancy between PowerBlot analysis data (protein levels) and cDNA array data (mRNA expression) in untreated and docetaxel-treated HNSCC cells and proposed that this discrepancy is due to posttranslational changes in proteins. In addition, changes in mRNA and protein stability may also account for differences between mRNA and protein analyses.

Others have used proteomic-based approaches to study lung physiology and cancer. Two-dimensional PAGE was used to separate proteins expressed differentially between human lung squamous carcinoma and surrounding normal bronchial epithelial tissue and MALDI-TOF MS and database searching were used to identify some of these proteins (16). The MALDI-TOF MS method has also been applied directly to frozen sections for profiling of protein expression in surgically resected normal and malignant lung tissues (18). The PowerBlot Western array complements these techniques because it is sensitive down to the subnanogram level, which cannot be accomplished for low-abundance proteins using two-dimensional PAGE/MS, and because the proteins' identities are well established by the corresponding antibodies.

Prominent among the proteins that are increased in the tumorigenic cells is the group linked to DNA synthesis and repair, which is expected from highly proliferative malignant cells (21). For example, proliferating cell nuclear antigen, a subunit of DNA polymerase {delta}, which increased in the 1170-I cells, is associated with DNA replication and with poor prognosis of NSCLC patients (26) and with resistance of lung cancer cells to chemotherapy and radiation treatment (27). The expression of MSH2 has been correlated with p53 overexpression in NSCLC samples (28). However, in the 1170-I cells p53 is inactivated by SV40 large T antigen; therefore, the increase in MSH2 protein is independent of p53 function.

Several of the proteins that show increased levels in the tumorigenic 1170-I cells had been reported to function as oncogenes. For example, STAT5 (29) has been found to activate cyclin D1, c-Myc, and Bcl-XL expression; to promote cell cycle progression and cellular transformation; and to prevent apoptosis (30). TEF-1 can promote immortalization and transformation (31). Some of the proteins found to decrease in the tumorigenic cells, such as CUL-2, Rb, E-cadherin, and {gamma}-catenin, are known to play a role as tumor suppressors, and their decreased expression may be related to the tumorigenic phenotype. The concurrent decrease in E-cadherin and {gamma}-catenin may decrease cell adhesion and contribute to increased malignant behavior (32).

The increases in components of the PI3K/Akt cell survival pathway (PDK1, Akt, CaMKK, p70S6K, and cyclin D3) as well as the decrease in the proapoptotic IGFBP3 are expected to have contributed to the tumorigenic phenotype in 1170-I cells. In contrast, the increase in the proapoptotic proteins Bax, Bid, and procaspase-7 and procaspase-8, which were detected by the PowerBlot and confirmed by Western blotting and immunohistochemistry (caspase-8), and in procaspase-3 observed only by Western blotting, are not easy to reconcile because they are not related to rates of spontaneous apoptosis measured by the terminal deoxynucleotidyl transferase–mediated nick-end labeling assay in the various normal, immortalized, transformed, and tumorigenic lung epithelial cells (data not shown). Malignant lung epithelial cells may be resistant to the proapoptotic caspases that they contain possibly as a result of high levels of antiapoptotic proteins (33).

The immortalized (BEAS-2B and 1799), transformed (1198), and tumorigenic (1170-I) cells (21) have been chosen because we surmised that a comparison of protein changes among these cells could provide a good starting point to identify markers for diagnosis and targets for intervention. The changes in protein levels among these cell lines indicate that the levels of some proteins changed "early" in this carcinogenesis model (i.e., between normal and immortalized cells), and this change was sustained through the tumorigenic state. The level of other proteins was changed progressively. We also analyzed protein levels in five cell lines derived from human NSCLC tumors. Only four cell lines (BEAS-2B, 1799, 1198, and 1170-I) showed the p53 protein band in Western blotting. This result could be expected because these cell lines were immortalized by SV40 large T antigen, which forms a complex with p53 to suppress its transcriptional activity (34) but, within the complex, p53 may be protected from degradation. The increase in cyclin B1 and CDK1 in immortalized and transformed cells (Fig. 3A) may be explained by the increased proliferation of these cells relative to NHBE, which results in a higher proportion of cells in the S and G2-M phases of the cell cycle. Others have reported that cyclin B1 expression in NSCLC tumors in vivo is associated with higher Ki-67 proliferative index and a poorer prognosis (35). Likewise, Eg5, a kinesin implicated in the formation of the bipolar spindle and its movement before and during anaphase, is essential for mitosis (36). Its levels increased in the immortalized, transformed, and tumorigenic lung epithelial cells (Fig. 2A). Interestingly, Eg5 has been implicated as an oncogene in the development of B-cell leukemias (37). Because Eg5 is phosphorylated by Cdc2 (CDK1)/cyclin B (38), the increase in its level may be related to the increase in both CDK1 and cyclin B1 observed in the same cells (Fig. 2). The increase in cyclin B1 was observed in the in vitro carcinogenesis model as well as in cell lines derived from human cancers (e.g., H460, H1792, SK-MES-1, and A549), in contrast with the increases in CDK1, Eg5, Annexin VI, and Bax, which were not observed in the NSCLC cell lines.

Studies with tissue specimens from human NSCLC tumors have found strong procaspase-3 protein by immunostaining had a poor 5-year survival (39). Caspase-8 has been detected in all 22 cases of NSCLC tissue samples analyzed in another study (40).

Our finding of an increase in the levels of Akt and the increases in the levels of the Akt downstream targets p70S6K and cyclin D3 (Fig. 2C) supports the contention that the PI3K/Akt pathway may be activated during the progression of cells to malignancy (41, 42). PI3K protein levels did not increase much; however, CaMKK, which can phosphorylate Akt on Thr308 (43), did show a gradual increase in the carcinogenesis model.

To begin to determine whether some of these protein changes occur in vivo, we used tissue microarrays prepared from resected human lung tissue specimens, which included normal, hyperplasia, squamous metaplasias, dysplasias, SCCs, AAHs, and adenocarcinomas for immunohistochemical analyses. To the best of our knowledge, this is the first time that a tissue microarray with so many premalignant lung tissue samples has been created and analyzed. In general, there was a good relationship between the changes observed in the levels of the four representative proteins (E-cadherin, caspase-8, p70s6K, and STAT5) detected by the PowerBlot, the conventional Western blotting, and immunohistochemical analysis. Many previous studies have compared levels of proteins in normal and malignant lung tissue samples by immunohistochemistry. However, our tissue microarray allowed us to compare for the first time the levels of proteins in different stages of SCC development, including low-grade dysplasia, high-grade dysplasias (including carcinoma in situ), and SCC. Interestingly, for E-cadherin, there was a progressive change (decrease) at different stages between normal and SCC, whereas the levels of nuclear STAT5 and p70s6K in SCCs were lower than in the high-grade dysplasias. In addition, our findings highlight the importance of validating immunoblotting blotting data obtained with cell or tissue lysates by immunohistochemistry using histologic sections because some proteins may be located in different cell compartments. For example, we found that STAT5 is located in both the nucleus and the cytoplasm, and its level in these two cellular compartments changes in a distinct fashion at different stages of SCC development. Specifically, the cytoplasmic STAT5 level is similar in SCC and high-grade dysplasia, and both are higher than levels in normal bronchial epithelial cells; in contrast, the level of nuclear STAT5 is higher in high-grade dysplasia than in normal cells but drops to normal levels in SCCs.

In conclusion, we have shown for the first time the usefulness of a high-throughput immunoblotting approach combined with an in vitro carcinogenesis cell model and tissue array to identify a variety of changes that occur in the level of different proteins involved in fundamental cellular processes potentially associated with lung carcinogenesis. Some of these proteins change early (between normal and immortalized cells), and others change late (between transformed and tumorigenic cells). They may serve as markers for diagnosis or prognosis and are potential targets for intervention in the carcinogenesis process by chemoprevention or therapy.


   Acknowledgments
 
Grant support: Early Detection Research Network UO1 grant CA86390 (Margaret Spitz, PI), National Cancer Institute Cancer Center Support grant P30 CA-16672-30, and Department of Defense grant DAMD17-01-1-0689 (BESCT, W.K. Hong, PI).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   Footnotes
 
Note: J. Shen and C. Behrens have contributed equally to this research and should both be considered first authors.

Current address for J. Shen: Department of Cancer Prevention and Population Sciences, Roswell Park Cancer Institute, Elm and Carlton Streets, Buffalo, NY 14263.

Received 4/23/04. Revised 5/27/06. Accepted 9/14/06.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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