Secretome-Based Identification and Characterization of Potential Biomarkers in Thyroid Cancer Lawrence Kashat,†,‡,§ Anthony K.-C. So,†,‡ Olena Masui,| X. Simon Wang,| Jun Cao,†,‡ Xianwang Meng,†,‡ Christina MacMillan,⊥ Laurie E. Ailles,# K. W. Michael Siu,| Ranju Ralhan,*,†,‡,§,|,⊥,¶,∇ and Paul G. Walfish*,†,‡,§,⊥,¶,∇ Joseph and Mildred Sonshine Family Centre for Head and Neck Diseases, Mount Sinai Hospital, 600 University Avenue, Toronto, Ontario, Canada M5G 1X5, Alex and Simona Shnaider Laboratory in Molecular Oncology, Department of Pathology & Laboratory Medicine, Mount Sinai Hospital, Joseph & Wolf Lebovic Health Complex, 600 University Avenue, Room 6-500, Toronto, Ontario, Canada M5G 1X5, Institute of Medical Science, University of Toronto, 7213 Medical Science Building, 1 King’s College Circle, Toronto, Ontario, Canada M5S 1A8, Department of Chemistry and Centre for Research in Mass Spectrometry, York University, 4700 Keele Street, Toronto, Ontario, Canada M3J 1P3, Department of Pathology & Laboratory Medicine, Mount Sinai Hospital, Joseph & Wolf Lebovic Health Complex, 600 University Avenue, Room 6-500, Toronto, Ontario, Canada M5G 1X5, Division of Stem Cell and Developmental Biology, Ontario Cancer Institute, MaRS Centre, Toronto Medical Discovery Tower, eighth floor 8-363, 101 College Street, Toronto, Ontario, Canada M5G 1L7, Department of Otolaryngology-Head and Neck Surgery, Mount Sinai Hospital, Toronto, Ontario, Canada M5G 1X5, Department of Otolaryngology-Head and Neck Surgery, University of Toronto, Toronto, Ontario, Canada M5G 2N2 Received May 27, 2010
In search of thyroid cancer biomarkers, proteins secreted by thyroid cancer cell lines, papillary-derived TPC-1 and anaplastic-derived CAL62, were analyzed using liquid chromatography-tandem mass spectrometry. Of 46 high-confidence identifications, 6 proteins were considered for verification in thyroid cancer patients’ tissue and blood. The localization of two proteins, nucleolin and prothymosin-R (PTMA), was confirmed in TPC-1 and CAL62 cells by confocal microscopy and immunohistochemically in xenografts of TPC-1 cells in NOD/SCID/γ mice and human thyroid cancers (48 tissues). Increased nuclear and cytoplasmic expression of PTMA was observed in anaplastic compared to papillary and poorly differentiated carcinomas. Nuclear expression of nucleolin was observed in all subtypes of thyroid carcinomas, along with faint cytoplasmic expression in anaplastic cancers. Importantly, PTMA, nucleolin, clusterin, cysteine-rich angiogenic inducer 61, enolase 1, and biotinidase were detected in thyroid cancer patients’ sera, warranting future analysis to confirm their potential as blood-based thyroid cancer markers. In conclusion, we demonstrated the potential of secretome analysis of thyroid cancer cell lines to identify novel proteins that can be independently verified in cell lines, xenografts, tumor tissues, and blood samples of thyroid cancer patients. These observations support their potential utility as minimally invasive biomarkers for thyroid carcinomas and their application in management of these diseases upon future validation. Keywords: proteomics • one-dimensional liquid chromatography-tandem mass spectrometry • secretome • thyroid cancer • biomarkers • secretory proteins
Introduction Thyroid cancer is the most common endocrine malignancy, with an estimated annual incidence of 122 800 cases worldwide * To whom correspondence should be addressed. Alex and Simona Shnaider Laboratory in Molecular Oncology, Department of Pathology & Laboratory Medicine, Mount Sinai Hospital, Joseph & Wolf Lebovic Health Complex, 600 University Avenue, Room 6-500, Toronto, Ontario, Canada M5G 1X5 and Department of Otolaryngology- Head and Neck Surgery, University of Toronto, Toronto, Ontario, Canada M5G 2N2. Tel: (416) 5864800 × 6426. Fax: (416) 586-8628. E-mail:
[email protected], rralhan@ mtsinai.on.ca. † Joseph and Mildred Sonshine Family Centre for Head and Neck Diseases, Mount Sinai Hospital. ‡ Alex and Simona Shnaider Laboratory in Molecular Oncology, Department of Pathology & Laboratory Medicine, Mount Sinai Hospital. § Institute of Medical Science, University of Toronto. | Department of Chemistry and Centre for Research in Mass Spectrometry, York University. ⊥ Department of Pathology & Laboratory Medicine, Mount Sinai Hospital. # Ontario Cancer Institute. ¶ Department of Otolaryngology-Head and Neck Surgery, Mount Sinai Hospital. ∇ Department of Otolaryngology-Head and Neck Surgery, University of Toronto. 10.1021/pr100529t
2010 American Chemical Society
and approximately 33 000 newly diagnosed cases in the U.S.A.1,2 There is a lack of molecular markers to predict the aggressiveness of thyroid cancers. Currently, fine-needle aspiration (FNA) is the most accurate preoperative technique for diagnosis of thyroid nodules. However, even using ultrasound-guided FNA, inconclusive biopsy results are quite common (10-20% of all cases).3 These patients normally undergo subsequent surgery to remove their thyroid glandsan invasive procedure that is often unnecessary as the majority of the suspected lesions are benign (>80%).4 Additionally, though most papillary thyroid cancers are nonaggressive and often nonmetastatic, a small percentage are in-fact aggressive and may develop distant metastasis leading to higher mortality.5 This establishes an urgent need for identifying biomarkers to distinguish benign thyroid nodules from malignant and aggressive carcinomas. The tumor cells and their interactions with the host’s microenvironment play vital roles in tumor growth, invasion, and metastasis.6 The cancer cells and the host’s microenvironment secrete and shed proteins or their fragments extracellularly and Journal of Proteome Research 2010, 9, 5757–5769 5757 Published on Web 09/27/2010
research articles into bodily fluids, including blood. These proteins and their fragments constitute the “cancer secretome”.7 Sampling of bodily fluids is minimally invasive, and multiple samples drawn over a period of time can provide longitudinal data during the course of disease investigation or treatment. In view of this, analyses of proteins in serum and saliva using mass spectrometry (MS)-based proteomic technologies have been carried out.8-10 Proteins secreted by cancer cells into their culture media (“secretome” proteins) make especially appealing targets for study because they may be detectable in bodily fluids.11-17 Herein we report the use of liquid chromatography-tandem mass spectrometry (LC-MS/MS) for secretome analyses of two thyroid cancer cell lines established from papillary thyroid cancer (TPC-1) and anaplastic thyroid cancer (CAL62). These cell lines may help identify proteins that human thyroid cancer cells secrete in vivo. The identified proteins can then serve as candidates for targeted analyses of serum/plasma in which sensitive and specific methodologies that are based on multiple reaction monitoring (MRM) MS are applied.18-21 To ascertain that the identified secretome proteins are indeed present in blood and as a prelude to MRM assaying, we have verified by Western blot analyses a panel of six proteins in the sera of thyroid cancer patients versus cancer-free individuals. In addition, to confirm that these proteins were secreted by thyroid cancer cells, we have independently verified the expression of these proteins in TPC-1 and CAL62 thyroid cancer cells and their conditioned media (CM) by Western blotting. Further, we determined the expression of two of these proteins in xenografts of TPC-1 cells in immunocompromised (NOD/ SCID/γ) mice and in 48 human thyroid cancer tissue specimens using immunohistochemistry. These strategies enabled identification of secreted proteins for large-scale validation and paved the way for development of minimally invasive markers for future clinical applications in thyroid cancer patients.
Materials and Methods Cell Lines. Two thyroid cancer cell lines, TPC-1 (derived from a human papillary thyroid carcinoma) and CAL62 (derived from a human anaplastic thyroid carcinoma) were used in this study.22,23 TPC-1 cell line was kindly provided by Dr. S. Jiang (The Ohio State University, Columbus, OH) and CAL62 by Dr. J. Knauf (Sloan-Kettering Institute, New York, NY) with permission from Dr. M. Santoro (Medical School, University “Federico II” of Naples, Naples, Italy). To ensure the problem of crosscontamination and misidentification of cell lines was avoided, short tandem repeat (STR) profiles of each cell line were determined to match those of the original thyroid-derived cell lines as reported in previous studies by Schweppe et al.24 and/ or in the American Type Culture Collection (ATCC) and German Collection of Microorganisms and Cell Cultures (DSMZ). Previously published studies with these cell lines have demonstrated the expression of thyroid specific genes in these cell lines confirming their thyroid origin.24,25 Cell Culture and Serum Free Media Collection. The workflow of this study is shown in Figure 1A. TPC-1 cells were propagated in 25 mL of RPMI-1640 containing 100 µg/mL streptomycin and 100 U/mL penicillin, 10% fetal bovine serum (FBS) and 1% nonessential amino acids in 150 mm dishes to about 65% confluence. CAL62 cells were propagated in 25 mL of Dulbecco’s Modified Eagle’s Medium (DMEM) with high glucose containing streptomycin, penicillin and 10% FBS. Cells were incubated at 37 °C in a humidified atmosphere of 5% CO2/ 95% air. The culture media were then aspirated and cells were 5758
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Kashat et al. washed three times with phosphate-buffered saline (PBS). Thereafter, cells were washed once with serum-free culture media that was collected as a time 0 h control. Cells were then incubated in the serum-free culture media for 48 h. Thereafter, the conditioned media were collected, centrifuged at 5000× g for 5 min at 4 °C, filtered using a 0.2 µm nylon filter, snap frozen and stored at -80 °C until further use. Media was collected from at least 15 plates. Trypan blue staining was performed following collection of the conditioned media at 24 and 48 h to estimate the number of dead cells. Since more than 98% cells were viable at 48 h, this time period was chosen for further study. Optimization of Cell Culture Conditions for Collection of Conditioned Media. Cells are routinely grown in cell-culture media containing fetal bovine serum; however, the high abundance proteins present in serum would interfere with the detection of secreted proteins. For this reason, cell culture conditions needed to be optimized for conditioned media collection. To avoid this interference, the cells were washed thoroughly four times (three times with PBS and once with serum-free media) and then grown in conditioned media for 48 h, allowing secreted proteins to accumulate. To limit cellular stress under these conditions, cells were only placed in serumfree culture media when they reached about 60% confluence. Protein Precipitation from Conditioned Medium and LC-MS/MS Analysis. Proteins were precipitated from the pooled conditioned media using 0.2% sodium deoxycholate (Sigma Aldrich, St.Louis, MO) and 10% trichloroacetic acid (Sigma Aldrich, MO) as described earlier.26 Following 2 h incubation on ice, the samples were centrifuged at 11 000× g for 30 min and washed twice with ice-cold acetone. The precipitated proteins were then dissolved in 50 mM NH4HCO3 buffer, pH 7.5. The protein concentration was determined using the Bradford assay (Bio-Rad, Hercules, CA). Protein samples were then heated for 1 h at 65 °C in the presence of 5 mM dithiothreitol, cooled to room temperature, and incubated in dark for 1 h with 10 mM iodoacetamide for alkylation. Sequencing grade trypsin (Promega, Madison, WI) at 1:20 (w/ v) in 50 mM ammonium bicarbonate was subsequently added and the samples were incubated at 37 °C overnight. The trypsin digested samples were then dried under vacuum and dissolved in 10 µL of 0.1% formic acid. Experiments were repeated twice and each set was analyzed separately using LC-MS/MS. Liquid Chromatography-Tandem Mass Spectrometry. The trypsin digested samples were analyzed using online LC-MS/ MS. The nanobore LC system (LC Packings, Amsterdam, The Netherlands) and mass spectrometer (QSTAR Pulsar, Applied Biosystems/MDS SCIEX, Foster City, CA) have been described by some of us earlier.11,27 One µL aliquot of the sample was loaded onto a C18 reverse-phase precolumn (LC Packings: 300 µm × 5 mm) and desalted before separation on an RP analytical column (75 µm × 150 mm packed in-house with 3-µm Kromasil C18 beads with 100 Å pores, The Nest Group). We used a nonlinear binary gradient: eluant A consisting of 94.9% deionized water, 5.0% acetonitrile, and 0.1% formic acid (pH 3); and eluant B consisting of 5.0% deionized water, 94.9% acetonitrile, and 0.1% formic acid for the separation. Eluant A was used to load the sample onto the C18 precolumn at a flow rate of 25 µL min-1. After 8 min, the C18 precolumn was switched inline with the reverse-phase analytical column; separation was performed at 200 nL min-1 using a 180-min binary gradient shown below.
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Potential Biomarkers in Thyroid Cancer
Figure 1. (A) Schematic for workflow of methods. (B) Mass spectrum of prothymosin-R (PTMA). MS/MS spectrum of the detected peptide for identification of PTMA by liquid chromatography-tandem mass-spectrometry.
time (min) B (%)
0 5
5 5
10 15
120 35
140 60
145 80
155 80
157 5
189 stop
MS data were acquired in information-dependent acquisition (IDA) mode with the Analyst QS 1.1 and Bioanalyst Extension 1.1 software (Applied Biosystems/MDS SCIEX). MS cycles comprised a TOF MS survey scan with a mass range of 400-1500 Da for 1 s, followed by five product-ion scans with a mass range of 80-2000 Da for 2 s each. The collision energy (CE) was automatically controlled by the IDA CE Parameters script. Switching criteria were set to ions with m/z g 400 and e1500, charge states of 2-4, and abundances of g10 counts. Former target ions were excluded for 30 s, and ions within a 6-Da window were ignored. Additionally, the IDA Extensions II script was set to “no repetition” before dynamic exclusion
and to select a precursor ion nearest to a threshold of 10 counts on every fourth cycle. Bioinformatics - SignalP and SecretomeP - Determination of Secretory Proteins. LC-MS/MS data were searched using the ProteinPilot software (Applied Biosystems, Foster City, CA) against a Celera human protein database (CDS KBMS 2004112009) containing 178239 protein sequences. The cutoff for significance used for this search was set for a score of 1.3, which corresponds to a confidence score of 95%. We used Signal Peptide Predictor (SignalP, http://www.cbs.dtu.dk/ services/SignalP3.0) to analyze the secretion features of identified proteins.28 SignalP uses amino acid sequences to predict the existence and location of signal peptide cleavage sites. SignalP determines the likelihood a protein is a signaling peptide using numerous artificial neural networks and hidden Journal of Proteome Research • Vol. 9, No. 11, 2010 5759
research articles Markov model algorithms to detect signal peptides in protein sequences. A protein is considered classically secreted if it receives a signal peptide probability g0.9. To identify nonclassical, or leaderless, protein secretion SecretomeP (http://www.cbs.dtu.dk/services/SecretomeP2.0) was used.29 SecretomeP uses a neural network that combines six protein characteristics to determine if a protein is nonclassically secreted. These characteristics include: the number of atoms, number of positively charged residues, presence of transmembrane helices, presence of low-complexity regions, presence of pro-peptides, and subcellular localization. A protein is considered nonclassically secreted if it receives an NN-score g0.5 (note: only proteins that were not considered classically secreted, that is, received SignalP scores 70%. Staining intensity was then also scored semiquantitatively as follows: 0, none; 1, mild; 2, moderate; and 3, intense. A total score was then obtained (ranging from 0 to 7) by adding the percentage positivity scores and intensity scores for each
Potential Biomarkers in Thyroid Cancer section. Box plots were used to determine the distribution of total score of membranous, nuclear, and cytoplasmic nucleolin and PTMA in thyroid cancers. Xenografts of Thyroid Cancer TPC-1 Cell Line in NOD/ SCID/γ Mice. NOD/SCID/γ (c)(null) mice, (SCID) mutation and interleukin-2Rgamma (IL-2Rgamma) allelic mutation (gamma(c)(null)), were originally generated by 8 backcross matings of C57BL/6j-gamma(c)(null) mice and NOD/Shi-scid mice.31 The breeding colony of these mice is maintained by the Ontario Institute of Cancer Research for use of its researchers (L.A.) in the University Health Network Max Bell Animal Facility, Toronto, Canada. Tumor xenografts of thyroid cancer cell line TPC-1 were established in these immunocompromised mice to evaluate the in vivo expression profiles of PTMA and nucleolin. One million TPC-1 cells in matrigel were implanted subcutaneously on the flanks of the mice and the animals were monitored for 4-6 months. The tumors appeared within 4-6 weeks; mice were sacrificed after 10-24 weeks, tumors were excised, fixed in formalin and embedded in paraffin. Tissue sections (5 µm) were cut, stained with hematoxylin and eosin and reviewed by the pathologist. Serial sections were used for immunohistochemical analysis of PTMA and nucleolin as described above.
Results Proteins Secreted by Thyroid Cancer Cell Lines TPC-1 and CAL62. A total of 154 nonredundant proteins were identified in the secretome of thyroid cancer cell lines, TPC-1 and CAL62. Only blood proteins (albumin and globulins) were identified in the 0 h controls, which were excluded from the list of identified proteins. The proteins that were identified based on one peptide were excluded from further analysis. Proteins identified with at least two peptides with g95% confidence were considered as high-confidence identifications (Supplementary Tables 1S and 2S, Supporting Information). In addition, PTMA was also considered as its identification was based on a 99% confidence peptide (Figure 1B) and has previously been reported to have an important role in human cancers.32-35 After applying the high-confidence threshold to the identified protein list, 46 proteins remained as candidates for further analysis (Table 1). The detailed list of these 46 highconfidence proteins and their identification in each LC-MS/ MS analysis is provided in Supplementary Table 3S (Supporting Information). Eighty percent of the high-confidence proteins were identified in at least two of the four separate analyses. Literature searches revealed that 31 of the high confidence proteins have not yet been reported in thyroid cancer. A comparative analysis between these 46 high-confidence proteins and their identification in other cancer secretome data sets is available in Supplementary Table 4S (Supporting Information). In both cell lines, investigation into the reported localization of the identified proteins using Ingenuity Pathway Analysis revealed that membrane and extracellular proteins were predominantly detected (Supplementary Figure 1S, Supporting Information). Similarly, the reported functions of the identified proteins suggest many are involved in metabolic processes and signal transduction pathways (Supplementary Figure 2S, Supporting Information). It is important to note that this information is based on models generated by IPA on the basis of updated database knowledge and has not been experimentally verified. It should be considered with caution, but provides useful information about the potential role of the proteins identified by secretome analysis. In total, 17 proteins
research articles were identified in the conditioned media from both cell lines, 18 were found only in TPC-1, and 11 proteins only in CAL62 (Table 1). The majority (40/46) of these high-confidence identifications were deemed secretory according to their SignalP and SecretomeP scores. Six of these 46 high-confidence protein identifications were considered for further verification based upon their known biological functions and potential associations with human cancers.32-48 Fluorescence Microscopy of Nucleolin and PTMA in TPC-1 and CAL62 Human Thyroid Cancer Cells. The subcellular localization of PTMA and nucleolin was determined in TPC-1 and CAL62 thyroid cancer cells (Figure 2A and B). PTMA was found in the cytoplasm and nuclei of both cell lines, while nucleolin was detected in the nuclei of TPC-1 and CAL62 cells (Figure 2A and B). Xenografts of TPC-1 Thyroid Cancer Cells in NOD/SCID/ γ Mice Exhibit Protein Expression Pattern Similar to Cultured TPC-1 Thyroid Cancer Cells. Expressions of PTMA and nucleolin were determined in TPC-1 human-mouse xenografts (Figure 2C). PTMA was detected in the nucleus and cytoplasm of tumor cells, while nucleolin expression was mainly nuclear, confirming similar pattern of expression of these proteins in cultured thyroid cancer cells and tumor xenografts. Detection of Secretome Proteins in Human Sera, TPC-1 and CAL62 Thyroid Cancer Cells and their Serum Free Media by Western Blotting. We analyzed 17 sera from thyroid cancer patients and 9 cancer-free individuals to determine if a panel of six proteins, nucleolin, PTMA, clusterin, CYR61, biotinidase and enolase 1, could be detected in circulation of thyroid cancer patients (Figure 3A and Supplementary Figure 3S, Supporting Information). In addition, as proof of principle, we independently verified the detection of these proteins in the whole cell lysates and conditioned media of TPC-1 and CAL62 thyroid cancer cells (Supplementary Figure 3S, Supporting Information). These proteins have all been reported to have possible functions in thyroid and/or other cancers. Western blotting confirmed all these proteins to be present in TPC-1 and CAL62 cell lysates (Supplementary Figure 3S, Supporting Information). Additionally, all proteins were confirmed in the CM of TPC-1 and/or CAL62 cells, in accordance with their detection by LC-MS/MS (Supplementary Figure 3S, Supporting Information). Notably, all six proteins were detected in the thyroid cancer patient’s sera (Figure 3A and 3B). In aggreement with our immunohistochemical observations, PTMA was increased in thyroid cancer patients’ sera compared to normal individuals (Figure 3B). Interestingly, clusterin, enolase 1, and biotinidase were reduced in the sera of many thyroid cancer patients compared to the normal sera (Figure 3A and 3B). A negative control using goat antirabbit secondary antibody failed to detect any immunoreactive proteins. A 62 kDa protein was detected using a goat antimouse secondary antibody but at considerably lower levels than the bands detected when mouse primary antibodies were used. Immunohistochemical Analysis of PTMA and Nucleolin in Human Thyroid Cancer Tissues. The expression of nuclear PTMA was observed to increase in ATC compared to poorly differentiated (insular) and PTC (Figure 4A, Figure 5, and Supplementary Table 5S, Supporting Information). ATCs displayed markedly increaesd cytoplasmic PTMA staining, and increased expression was also observed in poorly differentiated carcinomas compared to PTC (Figure 4A, Figure 5, and Supplementary Table 5S, Supporting Information). Further, Journal of Proteome Research • Vol. 9, No. 11, 2010 5761
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Table 1. Proteins Identified in the Conditioned Media of TPC-1 and CAL62 Thyroid Cancer Cell Lines by Liquid Chromatography-Tandem Mass Spectrometry Analysis references
proteins 1 2 3 4 5 6 7 8 9
accession #
unique % coverage SignalP SecretomeP peptides Protein score (95) (>95 confidence) TPC-1 CAL62 Ontologya probabilityb probabilityc exosomes
Versican Clusterin V-type proton ATPase subunit S1 Cysteine-rich angiogenic inducer, 61 (CYR61) Gamma-glutamyl hydrolase Insulin-like growth factor-binding protein 7 Melanoma-Associated Antigen Metalloproteinase inhibitor 2
trm|Q59FG9 spt|P10909 spt|Q15904
5 19 4
2 24 7
5 12 2
* * *
trm|Q53FA4
4
6
2
spt|Q92820
6
15
spt|Q16270
4
19
Enolase 1
10 Stem cell growth factor 11 Syndecan-4 12 Metalloproteinase inhibitor 1 13 Tyrosine-protein kinase receptor UFO (AXL) 14 Agrin 15 Amyloid beta A4 protein 16 Amyloid-like protein 2 (APLP2) 17 Beta-2-microglobulin protein (B2M) 18 CD44 antigen
-
-
1.000
-
Wasenius et al.49
M
1.000
-
-
M
0.998
-
-
-
M M M
0.810 1.000 1.000
*
E
4
*
4
*
*
TC blood and/or tissue samples
0.449
trm|Q92626
5
2
3
*
C
0.923
spt|P16035
4
13
2
*
*
E
1.000
trm|Q53FT9
4
5
2
*
*
C
0.000
trm|Q5U0B9
5
16
4
*
E
0.996
Cheng et al.32 Yu et al.,62 Maeta et al.63 Cheruvanky et al76 -
spt|P31431 trm|Q5H9A7
8 5
25 64
4 7
* *
* *
E E
1.000 1.000
-
spt|P30530
6
5
3
*
*
E
1.000
-
spt|O00468 spt|P05067
25 11
8 11
11 6
* *
*
M M
0.003 1.000
trm|Q9BT36
4
9
5
*
*
C
trm|Q6IAT8
7
35
5
*
E
-
0.552
Komorowski et al.,64 Hawthorn et al.65 Sainaghi et al.50
1.000
Ghidoni et al77 -
Krause et al51 -
1.000
-
-
Jung et al.78,79 Ghidoni et al.77 -
-
Taylor et al.80 -
-
0.235
spt|P16070
2
3
3
*
M
0.997
19 Cystatin C
spt|P01034
4
19
3
*
*
M
1.000
20 Dystroglycan 21 Galectin-3-binding protein 22 Fibronectin 23 Nucleolin 24 Nucleophosmin
trm|Q969J9 spt|Q08380
2 2
4 2
7 9
* *
* *
M E
0.999 1.000
spt|P02751 spt|P19338 spt|P06748
2 13 2
25 13 10
36 6 2
* * *
E N N
1.000 0.000 0.000
25 Osteopontin
spt|P10451
2
15
3
*
E
1.000
26 Ubiquitin A-52 residue ribosomal protein fusion product 27 SET protein 28 Biotinidase 29 Lysyl oxidase-like 2 variant 30 Nidogen-1
trm|Q3MIH3
4
20
2
*
C
0.000
0.682
-
Fluge et al.52 -
trm|Q6FHZ5 spt|P43251 trm|Q53HV3
9 2 2
29 4 4
5 2 2
* * *
M E M
0.000 0.823 0.999
0.162 0.720
-
-
spt|P14543
4
4
2
*
C
1.000
4 15
5 23
2 9
* *
*
E E
1.000 0.999
*
31 Nucleobindin 1 32 Plasminogen activator, urokinase
33 Dickkopf-related protein 3 (DKK-3) 34 Thrombospondin 1 35 Calsyntenin-1 36 Basement Membrane Specific Heparan Sulfate Core Protein 37 Prothymosin-R (PTMA)d 38 Cadherin-2 (N-Cadherin) 39 Granulins (proepithelin) 40 Activated leukocyte cell adhesion molecule (ALCAM) 41 Peptidylproyl isomerase A (cyclophilin A)
5762
trm|Q53GX6 trm|Q5SWW9
trm|Q6PQ81
4
7
2
*
trm|Q59E99
34
14
15
*
trm|Q5UE58 spt|P98160
11 4
8 1
6 2
* *
trm|Q9NYD3
2
10
1
*
spt|P19022
3
3
2
spt|P28799
4
5
2
trm|Q1HGM9
2
4
2
trm|Q3KQW3
5
12
2
Journal of Proteome Research • Vol. 9, No. 11, 2010
*
0.386 0.813
Manetti et al.61 -
-
E
1.000
Fujarewicz et al.60 Al-Nedawi Horvatic Herceg et al.26 et al.,53 Chu et al.54 -
E
0.994
-
M E
1.000 1.000
-
N
0.000
*
M
0.999
-
Letsas et al33 -
*
E
0.999
-
-
*
M
0.985
-
-
*
C
0.001
-
-
*
0.701
0.339
-
Ghoneim et al.55 -
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Potential Biomarkers in Thyroid Cancer Table 1. Continued
references
proteins
accession #
unique % coverage SignalP SecretomeP peptides Protein score (95) (>95 confidence) TPC-1 CAL62 Ontologya probabilityb probabilityc exosomes 2
5
2
*
*
C
0.015
0.512
-
43 Cathepsin Z trm|Q5U000 44 Superoxide dismutase trm|Q6NR85
3 4
7 17
2 2
*
* *
O C
1.000 0.001
0.648
-
45 Putative uncharacterized protein 46 Insulin-like growth factor-binding protein 6 (IGFBP-6)
trm|Q8WVW5
7
11
3
*
C
0.000
0.494
-
spt|P24592
2
27
5
*
E
1.000
42 Vimentin
spt|P08670
-
TC blood and/or tissue samples Sato et al.,56 Watanbe et al.57 Lassoued et al.58 Aldred et al.59
a Ontologies of identified proteins were analyzed using IPA and GoMiner. C, cytoplasm; E, extracellular; M, plasma membrane; N, nucleus; O, other; U, unknown. b Signal peptides were predicted using the hidden Markov model of SignalP 3.0 (protein with SignalP probability g0.900 is considered secretory). c Nonclassical secretion of proteins was evaluated by the neural network output score of SecretomeP 2.0 (protein with SecretomeP probability g0.500 is considered secretory). d PTMA was identified from using 1 unique of 99% confidence (sequence: EVVEEAENGR, detected twice). Its observed precursor mass was 1130 Da, charge +2. No modifications were detected.
nearly all PTC cases exhibited minimal cytoplasmic PTMA expression. This establishes a trend whereby nuclear expression of PTMA is increased in ATCs compared to PTC and insular carcinomas. Cytoplasmic expression of PTMA was also increased in insular thyroid carcinomas compared to PTCs. Nuclear nucleolin expression was observed in all the thyroid cancer subtypes examined (Figure 4B, Figure 5, and Supplementary Table 6S, Supporting Information). Faint cytoplasmic staining of nucleolin was also observed in some of the ATC cases. Interestingly, while most regions of ATC tissues showed minimal cytoplasmic nucleolin expression, some areas exhibited strong cytoplasmic staining, occasionally accompanying a loss of nuclear nucleolin expression (Figure 4B); this was only observed in the ATCs.
Discussion Herein we demonstrated the potential of secretome analysis of thyroid cancer cell lines to identify secreted proteins that can be independently verified in cell lines, tumor xenografts, tumor tissues and blood samples of thyroid cancer patients. The majority of the 46 identified high-confidence proteins were deemed to be secretory according to their SignalP and SecretomeP scores, lending support to our strategy of finding secreted proteins using proteomic analysis of conditioned media of cultured thyroid cancer cells. Literature searches conducted on the identified proteins revealed that 31 of them have not yet been reported in thyroid cancer, demonstrating the ability of secretome analysis to reveal potential candidates for applicative consideration in thyroid cancer. In addition, 15 of these 46 proteins have been reported to be present in sera and/or tumor tissues of thyroid cancer patients. Of these proteins, 12 have been previously detected in thyroid cancer tissues: CYR61, melanoma-associated antigen, tyrosine-protein kinase receptor UFO (AXL), amyloid-beta A4 (APP), osteopontin, plasminogen activator urokinase, thrombospondin, PTMA, vimentin, superoxide dismutase, insulin-like growth factor binding protein 6 (IGFBP6), and nidogen 1.32,33,49-60 Cystatin C has previously been found in thyroid cancer patients’ sera,61 and both, matrix metalloproteinase inhibtor 1 and matrix metalloproteinase inhibtor 2 reported in thyroid cancer tissues and patients’sera.62-65 Our search of other cancer secretome data sets revealed that nearly all of high-confidence proteins
have been reported in the secretome data sets of breast cancers, pancreatic cancers, lung cancers, colorectal cancers, and/or nasopharyngeal cancers. This suggests the potential for many of these identified proteins to serve as biological markers in other epithelial cancers. One limitation of our current methodology stems from the use of one-dimensional liquid chromatography. Future approaches using more sensitive proteomic analysis such as iTRAQ-labeling and multidimensional LC-MS analysis is likely to increase the number of proteins identified.13,14,66,67 Although many of the proteins identified herein have been linked to cancer, 31 have not yet been reported in thyroid cancer. Our work verifying the expression and detection of some of these proteins in thyroid cancer tissues, patient blood samples, thyroid cancer cell lines, and xenografts provides researchers and clinicians with useful information about their potential clinical relevance. Our study examined the localization of two proteins, PTMA and nucleolin, in two thyroid cancer cell lines and compared it with the expression profile of xenografts of this cell line from NOD/SCID/γ mice. The detection of these proteins in the cultured cells and their conditioned media confirmed these secretome proteins originated from thyroid cancer cells. Notably, the subcellular localization of PTMA and nucleolin was similar in TPC-1 cells and their xenografts in NOD/SCID/γ mice. We independently verified the expression of six secretome proteins in sera of thyroid cancer patients, biotinidase, nucleolin, enolase 1, PTMA, CYR61 and clusterin based on their reported implication in cancer.32-48 Our study’s identification of these six proteins in the secretome of thyroid cancer cells and their subsequent verification in thyroid cancer patients’ sera demonstrates how analysis of secretome proteins can identify potential biomarkers for use in the creation of minimally invasive blood-based diagnostic tests in future studies. These six proteins have been discussed here indepth and warrant analysis in large-scale study of thyroid cancer patients’ sera to determine their potential as minimally invasive thyroid cancer markers. Notably, in addition to these six proteins with known roles in cancer, many proteins have been identified in our study whose role in cancer remains unclear and may now be investigated for future mechanistic studies as well as exploration of therapeutic and diagnostic potential. Journal of Proteome Research • Vol. 9, No. 11, 2010 5763
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Kashat et al. PTMA is a heterochromatin remodelling protein shown to be significantly elevated in well-differentiated thyroid carcinomas compared to ademonas and goitres.33 Our study revealed PTMA to be localized in either the nucleus, cytoplasm, or both, in human thyroid cancer tissues. This was similar to the localization of PTMA in the cultured thyroid cancer cell lines and xenografts of TPC-1 in NOD/SCID/γ mice. Our immunohistochemical analysis of PTMA expression in thyroid cancer tissues suggests its nuclear and cytoplasmic expression is elevated in ATC compared to PTC and poorly differentiated carcinomas, indicating it may serve as a marker for aggressive carcinomas upon validation in a larger study. Cytoplasmic expression of PTMA was also increased in poorly differentiated carcinomas compared to PTC, which showed minimal cytoplasmic PTMA staining. In support of our findings, PTMA’s nuclear and cytoplasmic expression has been found to be significantly correlated with high tumor grade in bladder cancer.35 The cytoplasmic expression of PTMA is indicative of increased risk for tumor recurrence in the residual urinary tract of patients who have undergone a nephroureterectomy for upper urinary tract transitional cell carcinoma.32 These findings in different cancers, suggest that PTMA may play a critical role in the progression of tumors and its functions should be further examined. The clinical significance of cytoplasmic PTMA in thyroid tumor aggressiveness warrants confirmation in a larger longitudinal study. Furthermore, PTMA was detected and appeared elevated in sera of thyroid cancer patients compared to cancer-free individuals, making it a potential serological and histological marker for thyroid cancers. Although the band observed using mouse anti-PTMA antibody is similar in size to the band detected using antimouse secondary antibody alone, the intensity of the band detected using the mouse antiPTMA antibody is much greater and also consistent with our immunohistochemical observations of markedly increased PTMA expression in thyroid cancer tissues. These observations, in addition to our verification of the detection of PTMA in the lysates and conditioned media of TPC-1 and CAL62 cells, and its expression in the cultured cells and xenografts lends strong support for its identification in the sera of thyroid cancer patients.
Figure 2. Determination of subcellular localization of PTMA and nucleolin in cultured TPC-1 and CAL62 cells and xenografts of TPC-1 cells in NOD/SCID/γ mice. TPC-1 and CAL62 cells were grown on a glass slide up to 60% confluence and incubated with a PTMA (red) or nucleolin (green) antibody. Cells were stained with DAPI (blue) to reveal nuclei and slides were examined. (A) Immunofluorescence micrograph shows nuclear and cytoplasmic localization of PTMA (red) in TPC-1 thyroid cancer cells and nuclear localization of nucleolin (green) (original magnification ×1000). (B) Immunofluorescence micrograph shows nuclear and cytoplasmic localization of PTMA (red) in CAL62 thyroid cancer cells and nuclear localization of nucleolin (green) (original magnification ×1000). (C) High doses of single cell suspensions of TPC-1 cells in matrigel (1 million cells) were implanted subcutaneously on the flanks of NOD/SCID/γ mice and animals were monitored for up to 6 months. Tumors were subsequently excised, fixed in formalin, paraffin embdedded and tissue sections were immunostained with PTMA or nucleolin antibody. TPC-1 xenograft tissue sections show nuclear and cytoplasmic expression of PTMA and nuclear expression of nucleolin in tumor cells (original magnification ×400). 5764
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To our knowledge, this is the first report on the identification of biotinidase, nucleolin, and enolase 1 in thyroid cancer secretome. Nucleolin is a nuclear protein involved in several cell cycle processes. It does not have a known classical secretory signal, but has been shown to localize on the plasma membrane of proliferating cells.37,68 In our study, nucleolin was observed in tumor nuclei in all subtypes of thyroid cancers. Importantly, cytoplasmic localization of nucleolin was observed in ATCs only, suggesting cytoplasmic expression may be associated with aggressiveness of these cancers. Nucleolin was found to localize in the nucleolus in TPC-1 and CAL62 cells and in xenografts of TPC-1 cells in NOD/SCID/γ mice. Similar nucleolar localization of nucleolin has been found in breast cancer tissues and cell lines.38 Notably antagonists to surface nucleolin have been shown to suppress tumor growth and angiogenesis, suggesting an important link between cell-surface nucleolin expression and tumor progression.36 Herein its detectability in patient blood samples supports its secretion in thyroid cancers and warrants analysis in larger numbers of thyroid cancer patients’ sera to evaluate its potential as a thyroid cancer biomarker. Enolase 1 appeared to be reduced in thyroid cancer patients’ sera. It has been previously shown to be upregulated in male
Potential Biomarkers in Thyroid Cancer
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Figure 3. Immunodetection of identified proteins in sera of thyroid cancer patients. Protein samples were prepared from the sera (50 µg) of cancer-free individuals and the sera (50 µg) of thyroid cancer patients with nonmetastatic PTC or metastatic ATC, FTC, or PTC (meta). (A) Western blot analysis confirms the detection of biotinidase, clusterin, and CYR61 in the sera of thyroid cancer patients. Clusterin and biotinidase were reduced in thyroid cancer patient sera compared to normal sera. A negative control using goat antirabbit secondary antibody failed to detect any immunoreactive proteins. (B) Enolase 1, nucleolin, and PTMA were all detected in thyroid cancer patient sera. PTMA appears to increase in the sera of thyroid cancer patients compared to the sera from cancer-free individuals, consistent with our observations from immunohistochemical analysis of thyroid cancer tissues. As with clusterin and biotinidase, enolase 1 appears to decrease in thyroid cancer patient sera compared to normal sera. A faint 62 kDa protein was detected using a goat antimouse secondary antibody alone, but at considerably lower levels than the bands detected when mouse primary antibodies were also used.
Figure 4. Immunohistochemical analysis of PTMA in human thyroid cancer tissues. Fixed tissue sections of PTC and its variants, insular (poorly differentiated) thyroid carcinoma, and ATC were immunostained with antibodies for PTMA (brown) or nucleolin (brown) and nuclei counterstained with hematoxylin (blue). (A) Analysis of PTMA expression reveals increased expression of PTMA in both the nucleus and cytoplasm of ATC tissue compared to insular carcinomas and PTC. Cytoplasmic expression of PTMA was low to nonexistent in the PTC cases examined. (B) Similar nuclear staining of nucleolin intensity is seen among the various subtypes. Only the ATC cases examined revealed cytoplasmic expression of nucleolin. A and B, original magnification ×400. Journal of Proteome Research • Vol. 9, No. 11, 2010 5765
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Figure 5. Box plot analysis of immunohistochemical scoring of PTMA and nucleolin. Tumor samples were scored based on percentage positivity and immunostaining intensity. Sections were scored as positive if epithelial cells showed immunoreactivity in the plasma membrane, cytoplasm, and/or nucleus when observed by an evaluator who was blinded to the clinical history and outcome. Percentage positive scores were assigned according to the following scale: 0, < 10% cells; 1, 10-30% cells; 2, 30-50% cells; 3, 50-70% cells; and 4, >70%. Staining intensity was also scored semiquantitatively as follows: 0, none; 1, mild; 2, moderate; and 3, intense. The total score (0-7) was obtained by adding the percentage positivity scores and intensity scores for each section. (A) ATC displayed elevated nuclear expression of PTMA compared to PTC and insular thyroid carcinomas. ATC cases displayed strikingly elevated cytoplasmic expression of PTMA compared to PTC and insular cells. PTC staining of cytoplasmic PTMA was minimal-to-absent. Insular (poorly differentiated) thyroid carcinomas also demonstrated an increased expression of cytoplasmic PTMA compared to PTCs. (B) Nuclear expression of nucleolin was seen in all thyroid subtypes examined. Faint cytoplasmic expression was also observed in ATC cases only.
breast cancer tissue of the infiltrating ductal carcinoma subtype.48,69 Herein we also report an apparent decrease in biotinidase levels in thyroid cancer patients’ sera. Our findings are supported by a similar decrease in biotinidase levels recently reported in sera and tumor tissues of breast cancer patients.47 In this study biotinidase discriminated breast cancer patients from normal subjects with 47.6% sensitivity and 90.5% specificity. Biotinidase is involved in the transport of biotin in the blood and its intestinal absorption, and has also been implicated as a regulator of histone biotinylation70 and gene expression. The suitability of both these proteins as bloodbased thyroid cancer biomarkers warrants further investigation. CYR61, also known as CCN1, belongs to the CCN family of proteins, initially identified as secretory proteins whose production is induced by oncogenes,71 and has been shown to promote cellular proliferation, angiogenesis, and differentiation.72 Paradoxically, while having demonstrated importance in cancer cell proliferation, it has also been shown to play an important role in the induction of apotosis.73 It has been shown to be reduced to less than 50% of its normal levels in PTC, but has been demonstrated to be essential to the proliferation of prostate cancer cells.44,49 CYR61 has been reported in thyroid cancer tumor tissues, but herein we demonstrate the presence of CYR61 in sera of thyroid cancer patients, suggesting the possibility of development of serum based immunoassays for investigation of the diagnostic and prognostic potential of this protein in future studies. The identification of clusterin in the thyroid cancer secretome illustrates the powerful ability of secretome analysis to guide researchers to proteins with critical importance in the development and progression of cancer. Clusterin is a het5766
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erodimeric protein involved in numerous cellular functions including lipid transport, complement inhibition, apoptosis, DNA repair and cellular differentiation.39,74 Its secreted form has been shown to promote cellular survival and resistance to chemotherapy and radiation therapy.41,74,75 While the pathways involved in clusterin action are still being elucidated, it has been suggested that clusterin serves as a ubiquitin binding protein that enhances the activity of the transcription factor NF-kappaB by increasing the degradation of I-kappaB.43 The overexpression of clusterin in tumors has also been correlated with unfavorable survival, lymph-node metastasis, tumor invasion and TNM stage in gastric cancers40 and impaired survival in ovarian cancer.42 Clusterin appeared to be markedly decreased in the sera of thyroid cancer patients compared to the sera of cancer-free individuals. As clusterin has not yet been reported in thyroid cancers, its potential for improving the diagnosis, management, and treatment of thyroid carcinomas should be further examined.
Conclusions Our detection of proteins in the sera of thyroid cancer patients demonstrates the feasability of using a proteomicsbased secretome anlaysis approach to identify potential minimally invasive biomarkers. We identified novel proteins for future consideration in the management and diagnosis of thyroid cancer, including biotinidase, nucleolin, and enolase 1, which have been verified in thyroid cancer patients’ sera and/or tissues. Notably, immunohistochemistry revealed increased PTMA expression in both nucleus and cytoplasm of ATC, compared to poorly differentiated carcinomas and PTC. Furthermore, cytoplasmic expression of nucleolin was observed
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Potential Biomarkers in Thyroid Cancer only in ATC tissues, suggesting a possible correlation with tumor aggressiveness. Analysis of larger numbers of ATCs and poorly differentiated thyroid cancers in future studies is likely to establish the clinical relevance of these markers. Quantitation of these proteins in the sera of thyroid cancer patients and characterization of their expression in thyroid cancer tissues may serve as the next step toward evaluating the suitability of these proteins as potential thyroid cancer biomarkers. Abbreviations: APLP2, amyloid-like protein 2; APP, amyloidbeta A4; ATC, anaplastic thyroid carcinoma; AXL, tyrosine protein kinase receptor UFO; CE, collision energy; CYR61, cysteine-rich angiogenic inducer 61 variant; DKK-3, dickkopfrelated protein 3; FITC, fluorescein isothiocyanate; FNA, fineneedle aspiration; FTC, follicular thyroid carcinoma; IDA, information-dependent acquisition; IGFBP6, insulin-like growth factor binding protein 6; IPA, Ingenuity Pathway Analysis; LC, liquid chromatography; MRM, multiple reaction monitoring; MS, mass spectrometry; MTC, medullary thyroid carcinoma; PBS, phosphate-buffered saline; PTC, papillary thyroid carcinoma; PTMA, prothymosin-R; sCLU, secreted clusterin; STR, short tandem repeats; TBST, tris-buffered saline tween 20; TOF, time-of-flight; TRITC, tetramethyl rhodamine isothiocyanate.
Acknowledgment. P.G.W. and R.R. gratefully acknowledge support from the Joseph and Mildred Sonshine Centre for Head and Neck Diseases, Alex and Simona Shnaider Laboratory in Molecular Oncology, Temmy Latner/Dynacare, and the Department of Otolaryngology-Head and Neck Surgery, Mount Sinai Hospital, University of Toronto. The financial support of this work from Mount Sinai Foundation of Toronto, Da Vinci Gala Fundraiser and the Mount Sinai Hospital Department of Medicine Research Fund is gratefully acknowledged. K.W.M.S. acknowledges funding from the Canadian Institutes of Health Research (CIHR), and infrastructural support from the Ontario Research and Development Challenge Fund and AB SCIEX. We thank Dr. J. Knauf, Sloan-Kettering Insititute, New York, New York with permission from Dr. M. Santoro for the gift of CAL62 thyroid cancer cell line and Dr. S. Jiang, Ohio State University, Columbus, Ohio for the gift of TPC-1 thyroid cancer cell line. Supporting Information Available: The sequences of the peptides, confidence, and number of times observed for proteins identified in the serum-free media of TPC-1 and CAL62 thyroid cancer cell lines (combined data), Supplementary Table 1S. The total number nonredundant proteins, their locations and function (according to Protein Pilot) identified in the conditioned media of TPC-1 and CAL62 cell lines, Supplementary Table 2S. The ontologies and biological functions of the proteins, as determined by IPA are also provided, Figure 1S and Figure 2S. Overlap of proteins identified in the cell lines are provided, Figure 3S. A comparative analysis between the 46 high-confidence proteins and their identification in other cancer secretome data sets is available in Supplementary Table 4S.The The reported scores of all tissue observed are provided in Supplementary Table 5S and Table 6S. This material is available free of charge via the Internet at http://pubs.acs.org. References (1) Reis, E. M.; Ojopi, E. P.; Alberto, F. L.; Rahal, P.; Tsukumo, F.; Mancini, U. M.; Guimaraes, G. S.; Thompson, G. M.; Camacho, C.; Miracca, E.; Carvalho, A. L.; Machado, A. A.; Paquola, A. C.; Cerutti, J. M.; da Silva, A. M.; Pereira, G. G.; Valentini, S. R.; Nagai,
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