LC-MS/MS Analysis of Ovarian Cancer Metastasis-Related Proteins Using a Nude Mouse Model: 14-3-3 Zeta as a Candidate Biomarker Yifeng He,† Xin Wu,† Xiaohui Liu,‡ Guoquan Yan,‡ and Congjian Xu*,†,‡ Municipal Key Laboratory for Diseases Related to Women’s Reproductive and Endocrine Systems, Obstetrics and Gynecology Hospital, Fudan University, Shanghai 200011, People’s Republic of China, and Institute of Biomedical Sciences, Fudan University, Shanghai 200032, People’s Republic of China Received April 19, 2010
Peritoneal implantation is the most common metastatic pattern of epithelial ovarian cancer, and the five-year survival rate of patients is dramatically decreased when large-scale peritoneal metastasis occurs. This study aimed to determine serum proteins that could be used to detect early peritoneal metastasis of ovarian cancer. The secreted (or shed) proteins of the ovarian cancer cell line SKOV-3 were analyzed using LC-MS/MS, and 97 proteins were identified in the SKOV-3 culture supernatant. After the SKOV-3 cells were xenografted into the peritoneal cavities of nude mice, 3 of the 97 proteins were detected in animal sera. Following enzyme-linked immunosorbent assay (ELISA)-based screening of clinical blood samples, one of the three proteins, 14-3-3 zeta, was identified as a candidate biomarker. The average serum levels of 14-3-3 zeta in patients with epithelial ovarian cancer and benign gynecological diseases were significantly different. The expression of 14-3-3 zeta was associated with the degree of cancer peritoneal metastasis, the emergence of ascites, bilateral involvement, and the clinical stage and substage. Using 14-3-3 zeta, the overall diagnostic accuracy for ovarian cancer was greatly improved. Furthermore, siRNA-based experiments demonstrated that 14-3-3 zeta was responsible for approximately 62, 65, and 30% of the migratory, invasive, and implantation abilities of SKOV-3 cells, respectively. The present results demonstrated that the nude mouse xenograft model is an efficient system for performing function-oriented biomarker discovery, which can be used for a variety of research tasks in future molecular diagnoses, targeted therapies, and ovarian cancer vaccine development. Keywords: epithelial ovarian cancer • peritoneal metastasis • serum biomarker • nude mouse xenograft model • 14-3-3 zeta
Introduction Epithelial ovarian cancer (EOC) is a major gynecological malignancy, with an incidence rate of 3-12/100 000 woman/ year.1 In patients with Stage I ovarian cancer, the five-year survival rate is relatively high and can be greater than 90% because at this stage, the cancer has not yet invaded important pelvic and abdominal organs (such as the intestine and liver). When large-scale or remote metastasis occurs, the opportunity for long-term survival is dramatically reduced to a range of 2-80% (Stage II-IV).2,3 However, the ovary is located in the deep pelvic cavity, which provides sufficient space for tumor growth; therefore, early symptoms of ovarian cancer are often atypical or absent. The current method for detecting ovarian cancer is the joint use of pelvic ultrasonography and the serum CA125 test, which has a sensitivity and specificity of 70-80%.4-6 But under ultrasonography, most early ovarian cancers can resemble a benign tumor and are difficult to identify, even with * To whom correspondence should be addressed. Congjian Xu, Obstetrics and Gynecology Hospital, Fudan University, 419 Fanxie Road, Shanghai 200011, P. R. China. Tel: 86-21-63455050. Fax: 86-21-63450944. E-mail:
[email protected]. † Obstetrics and Gynecology Hospital. ‡ Institute of Biomedical Sciences.
6180 Journal of Proteome Research 2010, 9, 6180–6190 Published on Web 09/25/2010
the assistance of the serum CA125 test. At this stage, ovarian cancer is often confused with many benign diseases, including ovarian serous cystadenoma, endometriosis, and pelvic inflammatory disease (PID), which can also produce elevated CA125 serum levels. Ovarian cancer may not be diagnosed until ascites and intestinal obstructions form, but these changes are late events in cancer metastasis. Considering that the predominant biological characteristic that differentiates malignant from benign tumors is the ability to metastasize, it is necessary to identify metastasis-related serum biomarkers. These biomarkers could aid in the detection of ovarian cancer during its occult metastatic stage and in the early differential diagnosis of an unknown pelvic mass, which can result in a better prognosis for ovarian cancer patients. In the clinic, the most common metastatic pattern of ovarian cancer is peritoneal implantation.7 Although early peritoneal metastasis sites are usually too small to be detected macroscopically, they are fundamental for the development of epithelial ovarian cancer from Stage I to Stage II.8 However, proteomic changes in the serum that occur during cancer peritoneal metastasis are often transient, which makes it difficult to search for serum biomarkers in blood samples of early ovarian cancer patients. Patients who are suspected or 10.1021/pr100822v
2010 American Chemical Society
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14-3-3 Zeta and Ovarian Cancer Peritoneal Metastasis
Figure 1. Experimental workflow for the discovery of epithelial ovarian cancer metastasis-related serum biomarkers in the nude mouse xenograft model.
confirmed to have a certain stage of ovarian cancer require immediate treatment, which further decreases the opportunity to obtain sufficient qualified blood samples for research. In addition, because the proteins contained in preserved samples will inevitably be degraded, establishing a permanent bank to collect sufficient eligible ovarian cancer patient blood samples for mass spectrometry (MS) analysis is problematic.9 Thus, to remove many of the technological obstacles to the MS-based discovery of epithelial ovarian cancer metastasis-related serum biomarkers, we established a nude mouse xenograft model. We mimicked the peritoneal metastatic behavior of epithelial ovarian cancer cells by using an artificial method and MS to search for and identify human ovarian cancer metastasisrelated proteins in nude mouse sera. All of the nude mice used in the experiments had identical genetic backgrounds10 and were raised under the same conditions. Moreover, the established cancer metastasis could be confined to a specific stage, and the entire process was reproducible. Here, we report the process of discovering human serum proteins in the nude mouse xenograft model that were associated with the peritoneal metastatic behavior of ovarian cancer cells. We utilized a one-dimensional high-performance liquid chromatography-coupled electrospray ionization tandem mass spectrometry (LC-MS/MS) technique to identify these proteins.11 The diagnostic efficacies of these peritoneal metastasisrelated proteins were seriatim examined among patients with epithelial ovarian cancers of different stages by the enzymelinked immunosorbent assay (ELISA). Following the ELISA screening, one candidate serum biomarker, 14-3-3 zeta, was identified. This protein showed the highest diagnostic value for the detection and discrimination of early peritoneal implantation of epithelial ovarian cancer among all of the obtained metastasis-related serum proteins in our study. We also successfully validated the biological functions of 14-3-3 zeta in the metastatic behavior of epithelial ovarian cancer cells in in vitro and in vivo experiments.
Materials and Methods Study Design. The study comprised five parts: (1) LC-MS/ MS analysis of secreted (or shed) proteins in the supernatant of the epithelial ovarian cancer cell line SKOV-3; (2) establishment of a nude mouse ovarian cancer xenograft model by intraperitoneal injection of SKOV-3 cells; (3) LC-MS/MS analysis of serum samples collected from SKOV-3 cell-xenografted nude mice and comparison of the serum proteins of xenografted nude mice, serum background proteins of control nude mice and secreted (or shed) proteins of SKOV-3 cells; (4) clinical diagnostic screening of metastasis-related serum biomarkers among patients with epithelial ovarian cancers or benign gynecological diseases; and (5) in vitro and in vivo validation of the metastasis-promoting effect(s) of the serum candidate biomarker(s) on epithelial ovarian cancer cells (Figure 1). Ovarian Cancer Cell Line Culture. The epithelial ovarian cancer cell line SKOV-3 was purchased from the American Type Culture Collection (ATCC) and maintained in McCoy’s 5A medium (Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (Invitrogen) at 37 °C in 5% CO2. When the SKOV-3 cell density reached 106-107 cells/flask (>90% confluence in 175-cm2 flasks), the cells were washed 3 times with phosphate-buffered saline and transferred into 20 mL of serum-free, phenol red-free McCoy’s 5A medium. Immediately after the medium was changed (0 h), the SKOV-3 cell culture medium was collected, and another 20 mL of serum-free, phenol red-free McCoy’s 5A medium was added to the flask. The culture medium was collected again after 72 h. The collected medium was centrifuged at 3000× g for 10 min to remove cellular debris. The obtained supernatant was mixed with methanol at a 1:4 ratio and incubated at -20 °C for 2 h to precipitate the secreted (or shed) proteins. Subsequently, the supernatant:methanol mixture was centrifuged at 3000× g for 30 min, and the protein pellet was harvested. The obtained pellet was air-dried at room temperature, resuspended in 100 Journal of Proteome Research • Vol. 9, No. 12, 2010 6181
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Table 1. Clinico-Pathological Characteristics of the 160 Patients Enrolled in This Study epithelial ovarian cancer (80 cases)
characteristics
Baseline characteristics 56.9 ( 5.5 3.3 ( 1.5 1.6 ( 0.9 4.2 ( 3.9 7 1
Age (years)a Graviditya Paritya Postmenopausal yearsa Oral contraceptive use for g3 monthsb Ovulation stimulant use for g3 monthsc
Postoperative clinico-pathological findings Clinico-pathological diagnosis Epithelial ovarian cancer 80 Ovarian cystadenoma 0 Ovarian endometrioid cyst 0 Pelvic inflammatory disease 0 Metastasis peritoneal cavity, lymph nodes, and remote organs 67 a
Two-sided Student’s t test.
b
P-value
56.3 ( 6.3 3.5 ( 1.2 1.5 ( 0.8 3.5 ( 4.8 13 0
0.555 0.094 0.452 0.395 0.151 1.000
0 40 25 15
-
0
-
c
Two-sided χ2 test. Two-sided Fisher’s exact test.
µL of H2O, and digested with trypsin (Sigma-Aldrich, Germany) at 37 °C overnight. Clinical Blood Samples from Patients. The blood samples of patients with epithelial ovarian cancer (80 patients) or benign gynecological diseases (80 patients) were collected with informed consent at the Obstetrics and Gynecology Hospital, Fudan University, Shanghai, China (Table 1). The study was approved by the ethics committee of the same hospital. The requirements for blood sample collection were as follows: (1) in the morning, 10 mL of blood was drawn from each participant with a psychologically quiet status and an empty stomach; (2) the participant had a normal temperature and no acute infectious disease; (3) the participant had no other systemic diseases or chronic diseases of important organs, such as coronary heart disease, liver cirrhosis, pulmonary tuberculosis, chronic nephritis, diabetes mellitus, gout or rheumatic disease. The collected blood samples were allowed to clot at room temperature for 2 h and were then centrifuged at 1000× g for 10 min to separate the sera. High-abundance serum proteins, including albumin and IgG, were eliminated using the ProteoExtract Albumin/IgG Removal Kit (Calbiochem, San Diego, CA). Protein sample concentration and desalting were performed with Amicon Ultra-0.5 mL Centrifugal Filters according to the manufacturer’s instructions (Millipore, Billerica, MA). The obtained protein samples were digested with trypsin (Sigma-Aldrich) at 37 °C overnight. Nude Mouse Xenograft Model. The 20 BALB/c nude mice (20-22 g) used in the ovarian cancer xenograft experiment were female and 6 months old, and the 20 mice (20-22 g) used as controls were matched for age and sex. SKOV-3 cells (107-108 per mouse) or 1 mL of saline was intraperitoneally injected into nude mice in the xenograft or control groups, respectively. The xenografted and control nude mice were reared in an integrated ventilated cage (IVC) system with specific pathogen-free (SPF) barriers, and the room temperature was set at 22 °C. In preliminary experiments, we established that most SKOV-3 cell implantation nodes would be less than 5 mm in diameter 2-3 weeks after xenografting. Therefore, to capture the early serum proteomic changes of ovarian cancer metastasis, we collected blood samples from the xenografted and control nude mice within 2-3 weeks after xenografting. Accordingly, 1 mL of blood was drawn from each nude mouse of both groups 15 days after intraperitoneal injection of SKOV-3 cells. Afterward, all of the 6182
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SKOV-3 xenografted mice were sacrificed, and their peritoneal metastasis sites were measured to verify maximal diameters of 50% of the MSidentified supernatant proteins in SKOV-3 cells. The transport, cytoskeleton and blood groups are the top three types of proteins and account for >50% of the MS-recognized serum proteins in xenografted or control nude mice. (B) Comparison of the three groups of proteins indicated that the three serum proteins (marked with asterisks) detected in the xenografted nude mice overlapped with the supernatant proteins of SKOV-3 cells and were different from those of control nude mice. These proteins are the ovarian cancer peritoneal metastasis-related proteins that were used as a pool for the further selection of eligible serum candidate biomarkers.
Results LC-MS/MS Analysis of Secreted (or Shed) Proteins in the Supernatants of SKOV-3 Ovarian Cancer Cell Cultures. The supernatant proteins of SKOV-3 ovarian cancer cells were analyzed by LC-MS/MS. To prepare the cell culture supernatants for MS analysis, SKOV-3 cells were grown in serum-free McCoy’s 5A medium until they reached 90% confluence. The supernatants of the ovarian cancer cell cultures were collected 0 and 72 h after the media were changed. In the LC-MS/MS analysis of the supernatants of SKOV-3 cell cultures, we removed the MS signals that represented residual fetal bovine serum proteins by comparing the 72-h proteomic components with the 0-h components. After three replicates of these LC-MS/MS analyses, a common set of 97 secreted (or shed) proteins was obtained, which accounted for 90.7% of the 107 proteins identified. They were classified according to the “biological process” domain GO terms and are shown in Figure 2A (also see Supplementary Tables 1 and 2, Supporting Information). LC-MS/MS Analysis of Metastasis-Related Proteins in the Sera of Xenografted and Control Nude Mice. The serum proteomic changes caused by secreted (or shed) proteins of SKOV-3 cells were investigated using the nude mouse xenograft model. One million SKOV-3 cells were injected into the peritoneal cavity of each nude mouse; 15 days later, blood samples were drawn from the nude mice. Their sera were mixed together and analyzed using LC-MS/MS. We obtained 6184
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a set of 13 human proteins from the sera of xenografted nude mice (as a common set, accounting for 92.8% of the 14 proteins identified in three analysis replicates). In the nude mouse controls, 8 background serum proteins were found (as a common set accounting for 88.9% of the 9 proteins identified in three analysis replicates). We classified these serum proteins according to the GO terms (Figure 2A and Supplementary Tables 3-6, Supporting Information) and compared the two groups of proteins. Six differentially expressed proteins were identified, apolipoprotein A-I precursor, 14-3-3 protein zeta, complement factor B precursor, actin alpha-2, brain acid soluble protein 1 and apolipoprotein A-II precursor. The serum levels of these proteins were upregulated after SKOV-3 cancer cell xenografting. Three of these proteins, 14-3-3 zeta, apolipoprotein A-I precursor, and actin alpha-2, were secreted (or shed) by SKOV-3 cells (Figure 2B). These three proteins are ovarian cancer metastasis-related proteins that were able to pass successfully through the cancer-blood barrier following release from SKOV-3 cells. Comparison of the Diagnostic Efficacies of MetastasisRelated Serum Proteins and Screening for Candidate Biomarkers. To screen for eligible candidate biomarkers among the three differentially expressed serum proteins (14-3-3 zeta, apolipoprotein A-I precursor and actin alpha-2), we compared their respective diagnostic efficacies among 80 patients with epithelial ovarian cancer (Stage I-IV, 20 patients for each stage) and 80 patients with benign gynecological diseases by ELISA.
14-3-3 Zeta and Ovarian Cancer Peritoneal Metastasis
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Figure 3. Clinical diagnostic test results for the MS-identified metastasis-related serum proteins. (A) Serum levels of 14-3-3 zeta, apolipoprotein A-I (APOA-1) precursor and actin alpha-2 (ACTA2) in patients with epithelial ovarian cancer (EOC) or benign gynecological diseases (Benign) are shown. In each data set, the horizontal bars indicate the average serum levels of the index protein in the ovarian cancer patient and in the control groups. (B) General and stage-specific ROC curves of CA125, 14-3-3 zeta, APOA-1, and ACTA2 (left) and RMI-1 and RMI-2 (right) are indicated. According to the area under the ROC curve (general, stage-specific), the ranking of the clinical diagnostic ability of serum proteins was CA125 > 14-3-3 zeta > APOA-1 precursor > ACTA2. As compared with late-stage ovarian cancer (Stage III/IV), 14-3-3 zeta had a more significant effect on the improvement of clinical diagnostic performance in Stage I/II ovarian cancer patients. In addition, with the assistance of 14-3-3 zeta, both general and Stage I/II RMI-2 demonstrated better diagnostic performance compared to RMI-1.
The baseline characteristics and postoperational pathological findings for the 160 enrolled patients are presented in Table 1. The results showed that 14-3-3 zeta expression was significantly increased in patients with epithelial ovarian cancer compared to those with benign gynecological diseases (Figure 3A). The split-point analysis indicated that 14-3-3 zeta reached its optimal diagnostic efficacy with a cutoff value of 80 U/mL. At this value, the general sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) were 95, 82.5, 84.4, and 94.3%, respectively, and for Stage I/II patients, the parameters were 90, 82.5, 72, and 94.3%, respectively (Table 2). The ROC comparison showed that the diagnostic efficacy of 14-3-3 zeta was superior to that of CA125, especially for Stage I/II patients (Figure 3B). With respect to cancer pathological characteristics, the elevated serum level of 14-3-3 zeta was tightly associated with the emergence of ascites, bilateral cancer, degree of peritoneal metastasis, and clinical stage and substage (for Stage I), but it was not related to the age of the patient or the histological type of ovarian cancer (Table 3). Compared to CA125, the serum levels of 14-3-3 zeta were more significantly upregulated when microscopic peritoneal metastasis of the ovarian cancer occurred (P < 0.001) or when bilateral ovaries were involved (P ) 0.002), which implied that it could aid in differentiating Stage Ib and Ic ovarian cancers from Stage Ia ovarian cancers in the clinic (FIGO 1986 staging system).
Meanwhile, considering that the RMI system is a tool for incorporating different diagnostic methods to detect epithelial ovarian cancer, we combined 14-3-3 zeta, CA125, pelvic ultrasonography and menopausal status into a modified RMI: RMI-2 () 14-3-3 zeta × CA125 × US × M). The area under the ROC of RMI-2 was larger than that of the original RMI (RMI-1 ) CA125 × US × M) (0.973 vs 0.957, Figure 3B), which indicated that RMI-2 performed better overall compared to RMI-1. The Youden’s index of RMI-2 reached its highest value when the cutoff value was set at 12 000; at this value, the sensitivity, specificity, PPV and NPV of RMI-2 (general) were 92.5, 93.8, 93.7, and 92.6%, respectively (Table 2). In vitro Validation of the Metastasis-Promoting Effect of 14-3-3 Zeta on SKOV-3 Ovarian Cancer Cells. The LC-MS/ MS results obtained from the nude mouse model implied that 14-3-3 zeta might play a role in the metastasis of xenografted ovarian cancer cells. Thus, to obtain direct evidence of the metastasis-promoting effect of 14-3-3 zeta, we investigated the biobehavioral changes of SKOV-3 ovarian cancer cells before and after suppression of the 14-3-3 zeta gene. Seventy-two hours after the 14-3-3 zeta gene-specific siRNA was transfected, the mRNA and protein levels of 14-3-3 zeta in SKOV-3 cells were both reduced (Figure 4A). Next, using a 24-well Boyden chamber-based assay, we found that the 14-3-3 zeta genesuppressed SKOV-3 cells (migration: 15.3 ( 3.5%, invasion: 13.3 ( 2.5%, n ) 3) demonstrated 26% and 24% decreases in their Journal of Proteome Research • Vol. 9, No. 12, 2010 6185
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Table 2. Diagnostic Performances of the Serum (Candidate) Biomarkers and RMI Systems CA125 (g35 U/mL)a
items
d
apolipoprotein A-I precursor (g330 U/mL)b
14-3-3 zeta (g80 U/mL)b
Clinical diagnosis (Cases) 76 50 36 10 40 40 14 13 7 6 6 5 1 2
Epithelial ovarian cancer (80) Early stages (I/II, 40) Late stages (III/IV, 40) Benign diseases (80) Ovarian cystadenoma (40) Ovarian endometrioid cyst (25) Pelvic inflammatory disease (15)
68 28 40 29 17 9 3
Generalc I/IId III/IVe
Diagnostic 48.8% 33.8% 63.8%
accuracy (Youden’s index) 77.5% 63.8% 72.5% 8.8% 82.5% 83.8%
actin alpha-2 (g195 U/mL)b
RMI-1 (g150)a
RMI-2 (g12 000)b
56 18 38 23 11 10 2
69 29 40 10 5 4 1
74 34 40 5 3 2 0
41.3% 16.3% 66.3%
73.8% 60% 87.5%
86.3% 78.8% 93.8%
a Cut-off values set according to the literature.4-6 b Cut-off values set by the split-point method.13 c “Epithelial ovarian cancer” vs “Benign diseases”. “Stage I/II epithelial ovarian cancer” vs “Benign diseases”. e “Stage III/IV epithelial ovarian cancer” vs “Benign diseases”.
Table 3. Average Serum Levels of 14-3-3 Zeta According to the Clinical Characteristics of the Enrolled Patients with Epithelial Ovarian Cancer using CA12-5 as a Reference clinical characteristics (Cases)
Age 40-49 (5) 50-59 (47) g60 (28) Ascites No (42) Yes (38) Bilateral involvement No (29) Yes (51) Peritoneal metastasis site Negative cytological examination (19) Microscopic peritoneal metastasis site (11) Visible peritoneal metastasis site
