Patent Application
20110020846
The present invention relates to a cancer biomarker and an inspection method, particularly to an oral cancer biomarker and an inspection method using the same.
Oral cancer is the 11th most common human neoplasm in the world and is a complex disease arising in various organs, including tongue, buccal, hypopharynx, oropharynx, gum, palate, lips, and the floor of the mouth. Different parts of the tumor have distinct clinical presentations and outcomes, and are treated with different strategies. More than 90% of oral cancer cases are oral squamous cell carcinomas (OSCC), which are associated with a very poor prognosis. Previous studies have indicated the involvement of multiple genetic, epigenetic and metabolic changes in the evolution of OSCC, and these changes are strongly associated with environmental carcinogens such as tobacco, alcohol and betel quid chewing. In Taiwan, approximate 85% of OSCC patients have the custom of betel quid chewing, which has been suspected to be involved in the etiology of OSCC. Approximately 50-70% of OSCC patients die within 5 years of diagnosis, mainly due to local recurrence, metastasis to the esophagus or lungs, and/or the development of additional primary cancers. Late presentation, lack of suitable markers for early detection and failure of advanced lesions to respond to chemotherapy contribute to the poor outcome of this cancer. The overall 5-year survival rate and morbidity for patients with OSCC has not improved over the past two decades, and the World Health Organization predicts that the incidence of oral cancer will continuously increase worldwide, extending this trend into the next several decades.
Currently, OSCC is diagnosed through physical examination and excisional biopsies, and the treatment strategies rely on traditional surgery, radiotherapy, and chemotherapy. Radiologic or physical examination requires 1 to 2 cm of tumor mass for detection, and the clinical stages of OSCC determine the severity and prognosis of the cancer. Unfortunately, one study indicated that more than 50% of oral cancer patients in Taiwan presented with stage III or stage IV tumors. Despite the notable advantage of earlier diagnosis of head and neck cancers, and the fact that visual inspection or dye staining of the mouth can be useful for early detection of oral cancer and precancerous lesions, there is no currently accepted strategy for early diagnosis of OSCC.
Regarding biomarker research for OSCC, although studies have identified altered expression levels of many gene products in OSCC tissues, such gene products have yielded negligible definitive prognostic or predictive information to date. Recently, genomic (microarray) techniques have been used to identify the genes and molecular pathways involved in the progression of oral cancer, in an effort to support better classification of normal, pre-malignant and OSCC specimens, or improved prediction of patient outcomes. In contrast, relatively few studies have sought to systematically identify protein biomarkers for OSCC. Some studies have used 2D-gel protein profiling to identify proteins showing differential expression in OSCC tissue specimens. Subsequent protein identification using mass spectrometric analysis has led to the identification of approximately 40 proteins that are differentially expressed in OSCC tissues, but these proteins are not necessarily detectable in-accessible body fluids, such as plasma, serum or urine. For practical usage in tumor screening, biomarkers should be measurable in body fluid samples.
Thus, the present invention proposes an oral cancer biomarker and method for detecting oral cancer to overcome the abovementioned problems.
The primary objective of the present invention is to provide an oral cancer biomarker and an inspection method using the same, wherein the oral cancer biomarker can be directly detected in the specimen of the body fluid of a testee and thus can realize a fast and effective clinical diagnosis of oral cancer.
To achieve the abovementioned objective, the present invention proposes a biomarker for oral cancer diagnosis—Mca-2 binding protein (Mac-2BP), which is proved to exist in the body fluid of testees.
The present invention also proposes an oral cancer inspection method, which detects the Mac-2BP expression level of the body fluids of the testees suspected to have oral cancer.
The present invention further proposes an oral cancer inspection method using an oral cancer biomarker, which comprises steps: cultivating cell lines of oral cancer in a serum-free environment, and respectively collecting the proteins secreted by the cell lines; using 9-15% gradient electrophoresis to separate the proteins and staining the proteins with silver ion; cutting off the electrophoresis gel containing stained proteins, and using trypsin to hydrolyze in-gel proteins; using MALDI-TOF (Matrix-Assisted Laser Desorption/Ionization-Time Of Flight) to analyze the hydrolyzed proteins to identify the identities of the proteins respectively secreted by the cell lines; performing analysis to find out the proteins that have been identified in different cell lines simultaneously and using the proteins identified in different cell lines simultaneously as biomarkers.
Below, the embodiments are described in detail to make easily understood the objective, characteristics and accomplishes of the present invention.
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The present invention adopts Mac-2 BP binding protein (Mac-2BP) as a biomarker for detecting oral cancer.
Mac-2 BP is a secreted glycoprotein of 90-100 kDa, originally discovered as a tumor-associated antigen 90K (1, 2) and as a ligand of galectin-3 (formerly Mac-2) (3, 4). The functions of Mac-2 BP are not yet fully understood, although it is known to enhance cell-cell and cell-extracellular matrix adhesion (5) and induce production of IL-1, IL-6, and other cytokines from blood monocytes (4). Elevated expression levels of Mac-2 BP have been observed in tissues and sera of patients with different types of cancer, including breast cancer (2), non-Hodgkin's lymphoma (6), ovarian cancer (7), lung cancer (8), colon cancer (9) and NPC (10). Several evidences support that endogenous ligands of galectins including laminin, fibronectin, lysosome-associated membrane proteins and Mac-2 BP have been reported the altered expression in various cancer type were associated with patients clinical outcome. Mac-2BP was found as a tumor-associated antigen in human breast cancer originally and mostly expressed on the surface of tumor cells. It is synthesized and secreted in many cell type and serum level of Mac-2 BP in patient's peripheral blood have been found elevated in several human disease including infection by hepatitis B virus, hepatitis C virus (11), human immunodeficiency virus (12) and cancers. The level of high Mac-2 BP is associated with a poor prognosis (13-15). In a previously study of 310 patients with breast cancer, Mac-2 BP serum level was not correlated with tumor size, tumor histology or estrogen receptor status, but strongly associated with liver metastasis (16). Similarly, its expression was significantly associated with worse outcome and distant metastasis in stage I non-small cell lung cancers (17).
Although the following detailed description contains many specific details for the purposes of illustration, anyone of ordinary skill in the art will appreciate that many variations and alterations to the following details are within the scope of the invention. Accordingly, the examples of embodiments of the invention described below are set forth without any loss of generality to, and without imposing limitations upon, the claimed invention.
Identification of Proteins Released from the two OSCC Cell Lines
The inventor used a secretome-based strategy to identify potential OSCC biomarker(s) that might be detectable in body fluids such as serum or plasma.
Among the 17 proteins identified in both OSCC cell lines, 14 had been previously reported as being dysregulated in certain cancer types (Table 2). It is interesting to note that six of the 14 proteins (moesin, alpha enolase, fascin, glutathione s-transferase P, peroxiredoxin 1 and 14-3-3 zeta) were previously demonstrated as being overexpressed in oral cancer tissues in studies using immunohistochemistry, ELISA and/or Western blot analysis (Table 2). In addition, six proteins (heat shock protein 90, pyruvate kinase isozymes M1/M2, alpha enolase, glyceraldehyde 3-phophate dehydrogenase, triosephosphate isomerase and glutathione s-transferase P) were recently shown to be up-regulated in OSCC tissues by mass spectrometry-based proteomic approaches (21, 22, 23, 24). These observations suggest that identification of the proteins selectively enriched in the secretome of OSCC cell lines could be an efficient and convenient strategy for discovering proteins overexpressed in OSCC.
aSwiss-Prot accession numbers of identified proteins.
bNumbering of the protein bands corresponds to that in FIG. 1.
cMascot scores of proteins identified by peptide mass fingerprints.
dPercent sequence coverage (% seq cov) of matched peptides in the identified proteins.
eThe ontologies of identified proteins were analyzed using the Java application, GoMiner.
fThe signal peptides were predicted using the hidden Markov model of SignalP 3.0.
gThe nonclassical secretion of proteins was evaluated by the neural network output score of SecretomeP 2.0.
hThe protein has been reported to be present in exosomes.
iProtein identified in both cell lines are denoted in bold.
aDetection method: IHC, immunohistochemistry; Q-PCR, quantitative PCR; RT-PCR, reverse transcription-PCR; WB, Western blot; NB, Northern blot; 2DE, two-dimensional gel electrophpresis.
bReferences are denoted in Supporting Information.
To verify the mass spectrometry-based protein identification, conditioned media from the two OSCC cell lines were subjected to Western blot analysis for 15 selected targets, using antibodies available commercially or produced in the laboratory. The selected targets primarily consisted of proteins that had been shown to be dysregulated in at least one cancer type and were detected by mass spectrometry in the secretomes of the two OSCC cell lines; these included fibronectin, Mac-2 binding protein (Mac-2 BP), HSP90, moesin, ezrin, TGF beta-induced protein BIGH3, fascin, plasminogen activator inhibitor 1 (PAI-1), alpha enolase, phosphoglycerate kinase 1 (PGK1), glyceraldehyde 3-phophate dehydrogenase (G3PDH), fructose-bisphosphate aldolase A, glutathione s-transferase P (GST-pi), 14-3-3 zeta and cyclophilin A. Proteins (30 μg) from the conditioned medium of the two OSCC cell lines were resolved in 8 or 12.5% SDS gels, transferred to a PVDF membrane, and then probed with specific antibodies against the indicated target proteins. As shown in
Among the 15 proteins confirmed by Western blot analysis, the inventor chose the cell adhesion-related protein Mac-2 BP for further evaluation in terms of its clinical relevance in OSCC. Dysregulation of Mac-2 BP has been reported in many cancer types, but has not be investigated in OSCC. The inventor herein examined the expression of Mac-2 BP in 146 OSCC patients, using immunohistochemistry to test tissue specimens containing both tumor and non-tumor cells. The clinicopathological characteristics of the 146 OSCC patients enrolled in this study are shown in Table 3. The inventor detected positive Mac-2 BP staining of tumor cells in 111 (76.3%) cases, whereas only 43 (29.5%) cases showed positive staining of adjacent non-tumor cells (Table 4). Among the 111 cases that harbored Mac-2 BP-positive tumor cells, 72 cases (64.9%) were negative for Mac-2 BP expression in their adjacent non-tumor cells (Table 3). Among the 35 cases harboring Mac-2 BP-negative tumor cells, most (˜90%, 31 out of 35) showed adjacent non-tumor cells that were also negative for Mac-2 BP expression (Table 3). One representative case of positive Mac-2 BP staining in tumor cells is shown in
As mentioned, proteins upregulated in tumor tissues may or may not be detectable in accessible body fluids such as plasma and serum. However, proteins secreted by cancer cells could represent good serum/plasma biomarker candidates. The inventor previously developed a sensitive fluorimetric sandwich ELISA for Mac-2 BP that could be used to measure its level in blood samples (10). Here, the inventor used this method to examine whether the levels of Mac-2 BP were increased in the sera of OSCC patients versus healthy controls. The clinicopathological characteristics of the 88 OSCC patients and 106 healthy controls that provided serum samples in this study are shown in Table 5. In
Based on this finding, the inventor then examined the diagnostic efficacy of Mac-2 BP by receiver operating characteristic (ROC) curve analysis. The area under the ROC curve (AUC) was determined to be 0.72 (95% CI, 0.64-0.78) for Mac-2 BP (
OEC-M1 is an oral epidermal carcinoma cell line derived from the gingiva of a Chinese patient (25), whereas SCC4 is a tongue squamous cell carcinoma cell line derived from a 55-year-old male (ATCC No. CRL-1624). The two cell lines were grown in RPMI medium containing 10% fetal bovine serum (FBS), 25 mM HEPES and antibiotics at 37° C. in 5% CO2.
Harvest of Conditioned Media from Cancer Cell Lines
OEC-M1 and SCC4 cells were grown to confluence in 15-cm tissue culture dishes, and then washed twice with serum-free medium and incubated in serum-free medium for 24 hr. The conditioned media were harvested, centrifuged for elimination of intact cells, and concentrated by centrifugation in Amicon Ultra-15 tubes (molecular weight cutoff 5,000 Da; Millipore, Billerica, Mass.) three times at 4,000×g for 35 minutes each time. The concentrated samples were dried by Speed-Vac and resuspended in 100 μl deionized water for further use. The cells remaining on the dishes were washed twice with phosphate-buffered saline (PBS), and cell extracts were prepared as previously described (10, 26, 27). The protein concentrations of samples were determined using the BCA protein assay reagent from Pierce (Rockford, Ill.).
Proteins were resolved on 9-15% gradient SDS gels and subjected to silver staining, and images were captured using a Personal Densitometer SI (Molecular Dynamics, Amersham Biosciences, Piscataway, N.J.). Protein bands were excised, destained and subjected to trypsin digestion as previously described (10, 26). Briefly, gel pieces were destained in 1% potassium ferricyanide and 1.6% sodium thiosulfate (Sigma, St. Louis, Mo.), dehydrated in acetonitrile and dried in a SpeedVac. The proteins were then reduced with 25 mM NH4HCO3 containing 10 mM dithiothreitol (Biosynth AG, Staad, Switzerland) at 60° C. for 30 min, and alkylated with 55 mM iodoacetamide (Amersham Biosciences) at room temperature for 30 min. After reduction and alkylation, the proteins were digested with sequencing-grade modified porcine trypsin (20 μg/ml) (Promega, Madison, Wis.) overnight at 37° C. The resulting peptides were extracted with acetonitrile containing 0.1% trifluoroacetic acid (v/v), and loaded onto an MTP AnchorChip™ 600/384 TF (Bruker-Daltonik GmbH, Bremen, Germany). MALDI-TOF mass spectrometry was performed on an Ultraflex™ MALDI-TOF mass spectrometer (Bruker Daltonik GmbH, Bremen, Germany). Peptide mass fingerprints were acquired in reflectron mode (26.7 kV accelerating voltage) with 300 laser shots per spectrum.
Masses were automatically annotated using the Bruker Daltonics FlexAnalysis 2.2 software package (peak detection algorithm=SNAP; signal-to-noise threshold=2; maximal number of peaks=200; peak width=0.75 m/z; and quality factor threshold=50) and calibrated internally to a mass accuracy within 50 ppm, using a peptide mixture of bovine serum albumin (BSA) (m/z 927.49), human angiotensin II (m/z 1046.54), and ACTH-(18-39) (m/z 2465.198). Annotated and calibrated peaks were searched against the National Center for Biotechnology's non-redundant (NCBInr) database (released April 2005; 2,506,589 sequences and 850,049,330 residues) using the BioTools 2.2 software (Bruker Daltonics) and the Mascot search engine (version 2.1, Matrix Science, London, UK). Mascot searches were restricted to the human taxonomy (134,728 sequences), and with ‘trypsin digestion allowing a carbamidomethyl cysteine’ given as a fixed modification, and ‘oxidized methionine’ given as a potential variable modification. One trypsin-missed cleavage was allowed, and the peptide mass tolerance was set to 50 ppm. The known peptide masses of keratins were excluded. Positive identification was accepted when the data satisfied the following criteria: (i) targets were obtained with statistically significant search scores (greater than 95% confidence interval, equivalent to Mascot expected value <0.05); and (ii) the peptide ions of the identified proteins accounted for the majority of the ions present in the mass spectra. If the available peptides matched multiple members of a protein family in a Mascot search, the member with the highest ranked hit was selected. MS spectra with multiple matches were manually inspected to ensure the correct peptide-mass-fingerprint (PMF) assignment. Identified proteins were further analyzed using various software programs, including SignalP for predicting the presence of secretory signal peptide sequences (SignalP probability≧0.90)(28, 29), and SecretomeP for examining non-signal peptide-triggered protein secretion (SignalP probability<0.90 and SecretomeP score≧0.50)(30).
Anti-Mac-2 BP (120) and anti-heat shock protein 90 antibodies were produced in rabbits as previously described (10, 31). The antibody against BIGH3 was produced in rabbits using the peptide TQLYTDRTEKLRPEMEG(C), which corresponds to residues 118 to 134 of human BIGH3 (GenBank accession No. NM—000358). This peptide was synthesized by Kelowna International Scientific Inc. (Taipei, Taiwan). A cysteine residue was added to the C-terminus to facilitate coupling of the peptide to BSA (Sigma). The antibodies were produced and affinity purified according to previously described procedures (10).
The prepared samples (20 μg protein) were separated by SDS-PAGE, transferred to polyvinylidene difluoride (PVDF) membranes (Millipore), and then probed with various antibodies as previously described (10, 26). The utilized antibodies included anti-fibronectin (Santa Cruz Biotechnology, Santa Cruz, Calif.), anti-Mac-2 BP (120), anti-fascin (Santa Cruz Biotechnology), anti-heat shock protein 90, anti-moesin (Santa Cruz Biotechnology), anti-Ezrin (Abcam, Cambridge, Mass.), anti-BIGH3, anti-PAI-1 (Santa Cruz Biotechnology), anti-alpha enolase (Santa Cruz Biotechnology), anti-PGK1 (Santa Cruz Biotechnology), anti-G3PDH (Santa Cruz Biotechnology), anti-aldolase A (Santa Cruz Biotechnology), anti-GST-pi (Chemicon, Billerica, Mass.), anti-14-3-3 zeta (Upstate, Charlottesville, Va.), anti-cyclophilin A (Abcam) and anti-β-tubulin (MDbio, Taipei, Taiwan). Proteins of interest were detected with alkaline phosphatase-conjugated goat anti-rabbit IgG antibodies (Santa Cruz Biotechnology) and visualized with the CDP-Star™ chemiluminescent substrate (Boehringer Mannheim, Mannheim, Germany), according to the manufacturer's protocol.
Tumor specimens were obtained from 146 OSCC patients diagnosed at the Chang Gung Memorial Hospital (Tao-Yuan, Taiwan, Republic of China) in 1999-2000. The demographic data for these patients are shown in Supplementary Table S1. Serum samples were collected from 106 healthy controls [106 men ranging from 41 to 72 years of age (mean age 56.2±8.3)] and 91 OSCC patients [88 men and 3 women ranging from 29 to 74 years of age (mean age 49.1±10.8)] at the Chang Gung Memorial Hospital in 1999-2000. The enrolled cases included 5 stage-T1, 22 stage-T2, 11 stage-T3, 50 stage-T4 and 3 unknown stage patients. The study was approved by the Medical Ethics and Human Clinical Trial Committee at Chang Gung Memorial Hospital.
Tissue specimens were fixed with 10% formaldehyde, embedded in paraffin, and cut into 4-μm-thick sections. Staining for Mac-2 BP was carried out using the Envision-kit (DAKO Corp., Carpinteria, Calif.). The sections were deparaffinized with xylene, dehydrated with ethanol and then retrieved by boiling in 10 mM citrate buffer (pH 6.0) for 20 min. Endogenous peroxidase activities were inactivated with the Dual Endogenous Enzyme Block (DAKO Corp.) for 15 min at room temperature, and then the sections were blocked using the Antibody Diluent with Background Reducing Components (DAKO Corp.) for 30 min. The sections were incubated with rabbit polyclonal antibodies to Mac-2 BP (120) (50 μg/ml) overnight at 4° C., and then washed, and exposed to a peroxidase-conjugated secondary anti-rabbit antibody (DAKO Corp.) for 30 min at room temperature, followed by treatment with substrate-chromogen solution (DAKO Corp.) and a further incubation for 5-10 min at room temperature. Finally, the sections were counterstained with hematoxylin (DAKO Corp.), dehydrated and mounted. Immunohistochemistry (IHC) staining intensity and percentage were evaluated by a pathologist (Li-Yu Lee). The staining intensity was scored as 0 (no stain) or I (weak to strong), and the staining percentage was scored as 0 (0˜49%) or 1 (≧50%) (17). The two scores were multiplied by each other to get the final score. Positive staining was defined as a final score=1.
ELISA measurement of Mac-2 BP was performed as previously described (10). Briefly, white polystyrene microtiter plates (Corning, N.Y., USA) were coated with rabbit anti-Mac-2 BP (120) (10 μg/ml in PBS, 50 μl/well) antibodies overnight. The plates were then washed with TTBS and blocked with 200 μl of ovalbumin (Sigma) (1 mg/ml in TTBS). Recombinant MAC-2 BP (MedSystems Diagnostics GmbH, Vienna, Austria) was used as a standard. The serum samples comprised 0.5 μl of serum diluted to 50 μl in PBS containing 1% BSA were added and incubated at 37° C. for 1 h; the plates were then washed with TTBS. Subsequently, mouse anti-Mac-2 BP (BMS146, MedSystems Diagnostics GmbH, Vienna, Austria) (10 μg/ml in PBS, 50 μl/well) antibodies were applied and incubated for 1 h. After washing, 50 μl of alkaline phosphatase-conjugated goat anti-mouse IgG (Santa Cruz Biotechnology) (diluted 2000-fold in TTBS) was added and incubated for 1 h. Substrate 4-Methylumbelliferyl phosphate (100 μM; 100 μl/well, Molecular Probes, Eugene, Oreg., USA) was added, and then fluorescence was measured with a time-resolved fluorometer (the Plate Chameleon, Hidex, Turku, Finland) (λexcitation 355 nm, λremission: 460 nm).
The SAS® software package (version 8.2, SAS Institute, Cary, N.C.) was used to manage patient data and for statistical analysis. Mean between-group values were compared using the Chi-square or Fisher's exact tests. Wilcoxon Scores were used for ELISA group analysis. All statistical tests were two-sided, and p values less than 5% were considered significant. The receiver operating characteristic (ROC) curve was constructed by plotting sensitivity versus (1-specificity), considering each observed value as a possible cutoff value. The area under the ROC curve (AUC) was calculated as a single measure for the discriminative efficacy of each marker (32,33).
Those described above are only to exemplify the present invention but not to limit the scope of the present invention. Any modification or variation according to the spirit of the present invention is to be also included within the scope of the present invention, which is based on the claims stated below.
