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Clinical Cancer Research 13, 6842, November 15, 2007. Published Online First November 2, 2007;
doi: 10.1158/1078-0432.CCR-07-0569
© 2007 American Association for Cancer Research
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Cancer Susceptibility and Prevention

Annexin XI Is Associated with Cisplatin Resistance and Related to Tumor Recurrence in Ovarian Cancer Patients

Jin Song1, Ie-ming Shih1,2, Ritu Salani2, Daniel W. Chan1 and Zhen Zhang1

Authors' Affiliations: Departments of 1 Pathology and 2 Gynecology/Obstetrics and Oncology, Johns Hopkins Medical Institutions, Baltimore, Maryland

Requests for reprints: Zhen Zhang, Department of Pathology, Johns Hopkins Medical Institutions, 1550 Orleans Street, Baltimore, MD 21231. Phone: 410-502-7871; Fax: 410-502-7882; E-mail: zzhang7{at}jhmi.edu.


   Abstract
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 Abstract
Materials and Methods
 Results
 Discussion
 References
 
Purpose: Ovarian cancer patients treated with cisplatin-based chemotherapy often develop acquired cisplatin resistance and, consequently, cancer recurrence. The precise nature of chemoresistance remains unclear. In this study, a protein identified to be associated with cisplatin resistance in ovarian cancer cells was investigated in ovarian cancer tissues to address its clinical significance.

Experimental Design: Antibody microarrays were used to identify proteins consistently differentially expressed across three pairs of cisplatin-sensitive and cisplatin-resistant ovarian cancer cell lines. Immunoblotting was used to confirm observed alteration of protein expression. The protein expression was further evaluated by immunohistochemical staining using tissue microarrays containing various human normal and malignant tissues and 164 surgical specimens derived from primary and recurrent ovarian cancer patients who underwent primary debulking surgery followed by standard chemotherapeutic regimen.

Results: Annexin XI was down-regulated in all three cisplatin-resistant cell lines as compared with their parent cells. Annexin XI expression was observed in the majority of human normal organs and decreased in some of the most common human malignancies. The expression level of Annexin XI in first recurrent ovarian cancers was much lower than that in primary ovarian cancers (P = 0.0004). Increased Annexin XI immunoreactivity in ovarian cancers seemed to prolong the disease-free interval of patients (P = 0.03). Annexin XI immunoreactivity inversely correlated with in vitro cisplatin resistance in ovarian cancers (P = 0.01).

Conclusion: Decreased expression of Annexin XI is characteristic for cisplatin-resistant cancer cells and may contribute to tumor recurrence. Annexin XI may be a potential marker for chemoresistance and earlier recurrence of ovarian cancer patients.

Ovarian cancer is the fifth leading cause of cancer death among U.S. women and has the highest mortality rate of all gynecologic cancers (1). Due to the lack of effective screening tools and therapy, the mortality of ovarian cancer has not declined significantly in the past two decades. Most cases of ovarian cancer (~75%) are diagnosed at an advanced stage of the disease (1). Whereas patients with early-stage disease will have a >74% chance of survival, those with advanced-stage cancer will have overall survival rates of only 19% to 30% (1, 2). Administration of adjuvant chemotherapy consisting of a platinum compound (cisplatin or carboplatin) and a taxene remains the standard treatment for advanced-stage cancer following an optimal primary debulking surgery (3). One of the most important clinical problems in the treatment of ovarian cancer is the intrinsic or acquired resistance to cisplatin-based chemotherapy. Although it is initially very responsive (80%) to cisplatin-based chemotherapy, 75% of patients easily develop cisplatin resistance and relapse within 2 years of primary therapy (4). The progression of cisplatin-resistant cancer confers poor prognosis and decreases overall survival of this disease.

Several mechanisms such as decreased drug accumulation, enhanced detoxification, drug sequestration, faster repair of cisplatin-DNA adducts, and modulation of apoptotic pathways have been implicated in cisplatin resistance, but they are not sufficient to exhaustively explain this resistance emergence (5–9). Identification and characterization of more determinants of cisplatin resistance will advance our understanding of the varied mechanism that can contribute to this clinically relevant phenomenon and lead to the development of new protein markers or to the establishment of new therapeutic strategies. We hypothesize that among the unique array of differentially expressed proteins identified between chemotherapy-resistant and chemotherapy-sensitive ovarian cancer cells via novel proteomics technologies, some of them could be validated in ovarian cancer tissue samples. These proteins may be considered alone or in combination with others as a predictive marker of chemoresistance for platinum-based chemotherapy in patients with ovarian cancer.

Clontech Antibody Microarray 500 is a chip-based technology for simultaneous determination of relative abundance of hundreds of proteins in an analyte. The array is composed of more than 500 distinct monoclonal antibodies printed at high density on a glass slide in duplicate, which cover five major functional categories based on gene ontology: apoptosis, cancer, cell cycle, protein kinases, and neurobiology (10, 11). In this study, we used this high-throughput approach to identify proteins associated with acquired cisplatin resistance in human ovarian cancer cell lines. Proteins that were found consistently differentially expressed across multiple pairs of isogenic cisplatin-sensitive and cisplatin-resistant human ovarian cancer cell lines were further evaluated by immunohistochemical staining using tissue microarrays containing various human normal and malignant tissues and one panel of surgical specimens derived from primary and recurrent ovarian cancer patients who underwent optimal primary debulking surgery followed by a standard chemotherapeutic regimen.


   Materials and Methods
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 Abstract
 Materials and Methods
Results
 Discussion
 References
 
Cell lines and culture. A total of six human ovarian cancer cell lines (2008, 2008/C13*5.25, HEY, HEY C2, A2780, and A2780cis) were used in this study. Sublines stably resistant to cisplatin (2008/C13*5.25, HEY C2, and A2780cis) had been prepared from each parental cell line by repeated in vitro exposure to cisplatin as previously described (12). A2780 and A2780cis cells were purchased from European Collection of Animal Cell Cultures. 2008, 2008/C13*5.25, HEY, and HEY C2 cells were kindly provided by Dr. S.B. Howell (Department of Medicine, University of California-San Diego, La Jolla, CA). All cell lines were maintained in drug-free RPMI 1640 (Life Technologies/Invitrogen) supplemented with 10% (v/v) heat-inactivated fetal bovine serum (Hyclone) and 1% penicillin-streptomycin (Life Technologies/Invitrogen) at 37°C in a humidified atmosphere containing 5% CO2.

Cell cytotoxicity assay. The sensitivities of the cells to cisplatin were determined with a Cell Counting Kit-8 (Dojindo Molecular Technologies). Briefly, 100 µL of cell suspension (104 cells per well) were dispensed in 96-well microplates and incubated overnight at 37°C in a humidified incubator containing 5% CO2. Then the cells were treated with various concentrations of cisplatin diluted in 100 µL of conditioned medium (the final concentrations of cisplatin were 0, 1.56, 3.13, 6.25, 12.5, 25, 50, 100, and 200 µg/mL). After incubation for 48 h, 10 µL of Cell Counting Kit-8 solution were added to each well and the plates were further incubated for 4 h at 37°C. The absorbance at 450 nm was measured with a microplate reader (EL 312e Biokinetics reader, Biotek Instruments). Three independent experiments were done for each pair of cell lines.

Antibody microarray. Antibody Microarray 500 (Clontech Laboratories) was used to determine the protein expression profiles in three pairs of cell lines. According to the manufacturer's protocol, all antibodies3 have been extensively tested to verify their specificities. On average, the sensitivity range of the antibodies printed on the arrays is ~20 to 200 pg/mL of a given target protein.

Measuring protein abundances with Antibody Microarrays consists of five main steps: extracting protein, labeling protein with Cy5 and Cy3 dyes, removing unbound dye, incubating labeled protein with the arrays, and scanning arrays to measure bound antigen. Briefly, ~50 to 150 mg of cell pellets were thawed and homogenized in nondenaturing extraction/labeling buffer especially formulated and without protease inhibitors. The protein concentration of each extract was measured with the BCA Kit (Pierce Biotechnology). Each sample (e.g., sample A, drug-sensitive cell line; sample B, drug-resistant cell line) was then diluted to 1.1 mg protein/mL by adding the appropriate volume of extraction/labeling buffer. To control for differences in labeling efficiency, samples A and B were each split into two equal portions. Each portion was then labeled with either Cy5 or Cy3 (Amersham Biosciences) to produce four samples: A-Cy5, A-Cy3, B-Cy5, and B-Cy3 (i.e., 450 µL protein extract + 25 µL dye + 25 µL extraction/labeling buffer). After protein labeling, unbound dye was removed by gel exclusion chromatography (gel filtration) using a disposable PD-10 desalting column (Amersham Biosciences). Labeled proteins were collected by elution with 2.0 mL of 1x desalting buffer. Protein concentrations of labeled samples were determined using the BCA method. The average number of dye molecules covalently coupled to each protein (dye-to-protein ratio) was estimated following the manufacturer's protocol, which should be in the range of two to four. These four samples (50 µg/each) were combined to produce two mixtures (100 µg/each) of Cy5- and Cy3-labeled proteins: A-Cy5 is combined with B-Cy3 (mix 1), whereas A-Cy3 is combined with B-Cy5 (mix 2). The two mixtures (20 µg each) were then hybridized with the two identical Antibody Microarray slides in separate incubation chambers at room temperature for 30 min: slide 1 is hybridized with mix 1 and slide 2 is hybridized with mix 2. All steps were done as described by the manufacturer. The slides were washed, dried, and scanned under the optimized PMT settings using a GenePix 4000B scanner (Axon Instruments).

Grid alignment and initial quantification of array images were done using GenePix Pro 3.0 software. Signal intensities for each coordinate on the array were determined by background-subtracted median intensities from Cy5 and Cy3 channels. Array 1 measures A-Cy5/B-Cy3 (ratio 1) and array 2 measures B-Cy5/A-Cy3 (ratio 2). The derived Cy5/Cy3 ratios were imported into the Antibody Microarray Analysis Workbook (Microsoft Excel 97/98; Clontech Laboratories). An average internally normalized ratio (INR; the square root of average ratio 1 / ratio 2) is calculated, which represents the abundance of an antigen in sample A relative to that of sample B. Using the Antibody Microarray Workbook, average values (e.g., average ratio 1 / ratio 2 and average INR) are considered invalid if they are based on duplicates that differ by >30%. INR values are considered invalid if they are based on Cy5/Cy3 ratios in which one or more of the antigen signals are less than twice the background signal. As suggested by the manufacturer, INR values that are ≥1.3 or ≤0.77 indicate valid changes that signify differences in protein abundance. These threshold values were previously used successfully (11, 13). It is important to note that the threshold values of 1.3 and 0.77 are based on an ideal average INR of 1.0. However, an average INR of 1.0 is not normally obtained in actual experiments. Thus, threshold values should be calculated for each set of slides. The average INR for the experiment was then obtained by averaging the INR values of all antigens. The average INR for the experiment was multiplied by 1.3 to obtain the upper threshold value and by 0.77 to obtain the lower threshold value for that experiment. Proteins with INR values outside this interval were considered differentially expressed in the pair of cells. An INR greater than the upper threshold value indicates that an antigen is more abundant in cisplatin-sensitive cell line than in cisplatin-resistant cell line. Conversely, an INR less than the lower threshold value indicates that an antigen is less abundant in cisplatin-sensitive cell line than in cisplatin-resistant cell line. The protein consistently differentially expressed across multiple pairs of cells was subjected to further analysis.

Immunoblot analysis. All three pairs of cells were used to validate the above antibody microarrays results. Protein extraction and concentration measurement were done as described above. The denatured samples (40 or 80 µg of protein) of cisplatin-sensitive and cisplatin-resistant cell lines were electrophoresed on 4% to 15% gradient SDS-PAGE gels (Bio-Rad), electroblotted on nitrocellulose membranes (Bio-Rad), and probed with the same monoclonal antibody as printed on the arrays (anti–Annexin XI mouse immunoglobulin G1, clone 16, 1:10,000; BD Biosciences). The bound antibodies were visualized with horseradish peroxidase–conjugated secondary antibodies and enhanced chemiluminescence (Amersham Biosciences). Actin in the corresponding cell lysates was used as an additional control to show equal loading. Three independent experiments were done for each pair of cell lines.

Human subjects and tissue specimens. In accordance with the human subject research guidelines of institutional review board, formalin-fixed paraffin-embedded tissues were obtained from the Department of Pathology at Johns Hopkins Medical Institutions. These included 164 ovarian carcinoma tissues, which are 93 primary tumors, 57 first recurrent tumors, and 14 second or third recurrent tumors (Table 1 ). All patients underwent primary debulking surgery followed by platinum/paclitaxel-based combined chemotherapy. Among 57 first recurrent patients, 20 pairs of surgical specimens were derived from primary and matched recurrent lesions of the same patient. The tissue microarrays were constructed as described previously (14). Briefly, tissue cores (1.5 mm in diameter) were taken from three spatially separate areas in a single donor block from each case using a tissue microarrayer (Beecher Instruments). Cores were precisely arrayed into a recipient paraffin block at defined coordinates to form an array of 11 x 9 cores format. The selection of areas for cores and the confirmation of the presence of tumor on tissue microarrays were made by a pathologist (I-M.S.) based on review of the corresponding H&E slides from donor and recipient paraffin blocks. In addition, tissue arrays containing normal pancreas, kidney, stomach, colon, lung, bladder, prostate, uterus, placenta, breast, ovary, liver, cerebrum, testis, thyroid, thymus, and lymph node and arrays containing tumor tissues from cancers of the pancreas, kidney, stomach, colon, lung, bladder, prostate, uterus, breast, ovary, liver, cerebrum, testis, thyroid, lymph node, fibrous tissues, skin, and head and neck were obtained from US Biomax, Inc.


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  		Table 1. Clinicopathologic characteristics of the study cohort

 
Immunohistochemistry. The EnVision+ System-horseradish peroxidase (3,3'-diaminobenzidine; DakoCytomation) was used in this study. Four-micrometer-thick histologic sections were deparaffinized and rehydrated in graded ethanols. Antigen retrieval was done by placing slides in 10 mmol/L sodium citrate buffer (pH 6.0) inside a preheated steamer (95-100°C) for 25 min. Endogenous peroxidase activity was blocked with 0.03% hydrogen peroxide for 10 min. Sections were then incubated with the same monoclonal antibody as printed on the arrays (anti–Annexin XI mouse immunoglobulin G1, clone 16, 1:200; BD Biosciences) at room temperature for 40 min. After incubation, the specimens were washed with 1x TBST buffer and incubated with peroxidase labeled polymer at room temperature for 40 min. The samples were then washed with 1x TBST buffer and incubated with freshly prepared 3,3'-diaminobenzidine+ substrate-chromogen buffer at room temperature for 3 min. After gently rinsing with dH2O, slides were counterstained with hematoxylin and mounted with permanent mounting media. For negative controls, the primary antibody was omitted and replaced by the monoclonal mouse immunoglobulin G1 antibody to Aspergillus niger glucose oxidase, an enzyme which is neither present nor inducible in mammalian tissues (X0931, DakoCytomation).

Evaluation of immunohistochemical staining results. The immunohistochemical staining of the protein in the panel of ovarian carcinoma tissues was scored semiquantitatively from 0 to 3+ as follows: negative (0), weak (1+), moderate (2+), and strong (3+) expression. Briefly, both the percentage of stained cells (0, ≤10%; 1, 11-25%; 2, 26-50%; 3, 51-75%; 4, 76-90%; 5, ≥91%) and the intensity of the staining (0, none; 1, weak; 2, moderate; 3, strong) were assessed in every component on every slide as described previously (15, 16). A final score was obtained by combining the percentage of stained cells with the intensity of the staining as follows: samples with an intensity level of 0 or 1 in ≤10% of cells were designated negative, and samples with an intensity level of 1 in >10% of cells were designated weak. For intensity levels 2 and 3, combined scores of 2 to 3 were designated as weak, 4 to 6 as moderate, and 7 or 8 as strong expression. Each core on tissue microarrays was scored individually and then the final score for each case was determined by combining the results of replicate cores for that case. In addition, various normal tissues were considered positive for the protein expression if any staining was detectable. Various malignant tissues were considered positive for the protein expression if and staining was detectable in malignant cells (17).

Extreme drug resistance assay. In vitro drug responses of tumors were assessed by the extreme drug resistance assay (Oncotech; ref. 18). Briefly, fresh tumor tissue was minced and enzymatically digested to disaggregate the tumor cell, which were then plated in soft agar. Tumor cells were then exposed to antineoplastic agents (cisplatin, carboplatin, paclitaxel, and Taxotere) at a concentration of a maximum tolerated dose for 5 days in vitro. Tritiated thymidine was introduced during the last 2 days of culture as a measure of cell proliferation, and the drug-treated cells were compared with the untreated control. Assay results were divided into three categories: extreme, intermediate, and low drug resistance as described previously (18).

Statistical analyses. On the basis of available clinicopathologic information including age, stage, grade, histologic type, treatment history, recurrence status, survivorship, and extreme drug resistance status, Fisher's exact test was done to compare the protein differential expression levels in ovarian carcinomas. Survival curves were established using the Kaplan-Meier method, and their differences were analyzed using the log-rank test. Overall survival time was defined as the number of months between diagnosis and death. Disease-free interval was defined as the number of months between primary surgery and first tumor recurrence. Differences with P < 0.05 were considered statistically significant. All of the statistical analyses were done using Statistica 6.1 (Statsoft).


   Results
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 Abstract
 Materials and Methods
 Results
Discussion
 References
 
Sensitivity to cisplatin. Cell cytotoxicity assays were done to determine the sensitivities of the three pairs of cell lines to the cytotoxic effect of cisplatin. Dose-response curves were plotted on a semi-log scale as the percentage of the control cell number, which was obtained from the sample without drug exposure. Resistance to cisplatin in 2008/C13*5.25, HEY C2, and A2780cis cell lines was confirmed (P < 0.05; Fig. 1A ).


Figure 1
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  		Fig. 1. A, cell cytotoxicity assays. Resistance to cisplatin in 2008/C13*5.25, HEY C2, and A2780cis cell lines was confirmed (P < 0.05). B, validation of the antibody microarrays INR data by immunoblotting. The down-regulation of expression of Annexin XI was confirmed in all three pairs of cells. DR, drug-resistant cell line; DS, drug-sensitive cell line.

 
Determination of protein abundances in cisplatin-sensitive and cisplatin-resistant human ovarian cancer cells. Using antibody microarrays, we simultaneously measured expression levels of 500 proteins representing a broad range of functional classes in three pairs of cells. In general, 70% of antigens passed the filter criteria used in this study. A panel of proteins differentially expressed in three pairs of cells were identified for their potential associations with cisplatin resistance (Table 2 ). The respective threshold values of INR for three pairs of cells were as follows: 0.98-1.66 (2008 versus 2008/C13*5.25), 0.73-1.23 (HEY versus HEY C2), and 0.99-1.67 (A2780 versus A2780cis). Proteins with INR values outside the interval were considered differentially expressed in the pair of cells. Proteins with higher abundance in cisplatin-resistant cell lines were translin-associated factor X and nuclear domain 10 protein for 2008/C13*5.25; Na+/K+ ATPase b2 and caspase-7/Mch3 for HEY C2; and heat shock protein 60 for A2780cis. Proteins with higher abundance in cisplatin-sensitive cell lines were Annexin XI, 5-hydroxytryptamine 2A receptor/serotonin receptor, and multi-PDZ-domain protein 1 (MUPP1) for 2008; monocyte chemotactic protein 1, MUPP1, cyclooxygenase-2 (COX-2/PGHS), Annexin XI, mitogen-activated protein kinase 5, and TRAF2 and NCK-interacting protein kinase for HEY; and diacylglycerol kinase {theta} for A2780 (Table 2). Among these, Annexin XI and MUPP1 were consistently down-regulated in two cisplatin-resistant cell lines (2008/C13*5.25 and HEY C2) compared with their parent cell lines (2008 and HEY).


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  		Table 2. Determination of protein abundances in cisplatin-sensitive and cisplatin-resistant human ovarian cancer cells (2008 vs 2008/C13*5.25, HEY vs HEY C2, and A2780 vs A2780cis) using antibody microarrays

 
Validation of microarray data by immunoblotting. Annexin XI is a member of the Annexin superfamily of Ca2+-dependent and phospholipid-binding membrane-associated proteins implicated in Ca2+ signal transduction processes associated with cell growth and differentiation (19–22). Previously, Annexin IV has been found less expressed in the cisplatin-resistant cell lines (IGROV1-R10 and IGROV1/CP) in comparison with the sensitive parental cell line (IGROV1, another ovarian cancer cell line; refs. 23, 24). Therefore, we subsequently validated the alteration of Annexin XI in cisplatin-resistant ovarian cancer cell lines by immunoblotting. Immunoblotting with the same monoclonal antibody as printed on the array showed a single band corresponding to Annexin XI protein mass. Comparing with their parent cell lines, Annexin XI was found to be decreased not only in the two above-mentioned cell lines, 2008/C13*5.25 and HEY C2, but also in the third cisplatin-resistant cell line A2780cis (Fig. 1B). The amount of down-regulation of Annexin XI in three pairs of cells was estimated to be 3- to 8-fold using Quantity One 4.5 software (Bio-Rad).

Expression of Annexin XI in human normal and malignant tissues. To investigate the tissue expression pattern of Annexin XI, arrays containing various human normal tissues were stained with the same monoclonal antibody as used above. The experimental results showed that Annexin XI was expressed in majority of human normal organs including pancreas (11 of 12), kidney (10 of 11), stomach (8 of 9), colon (10 of 12), lung (9 of 13), bladder (4 of 5), prostate (9 of 10), uterus (5 of 5), placenta (2 of 4), breast (3 of 6), and ovary (2 of 4), but was not detected in liver (0 of 6), cerebrum (0 of 6), testis (0 of 4), thyroid (0 of 5), thymus (0 of 4), and lymph node (0 of 9). Examples of positive staining for Annexin XI in normal tissues (pancreas, stomach, colon, breast, kidney, and uterus) are presented in Fig. 2A (a-e and g).


Figure 2
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  		Fig. 2. A, immunohistochemical staining of normal and malignant human tissues for Annexin XI. a, normal pancreas; b, normal stomach; c, normal colon; d, normal breast; e, normal kidney; f, renal cell carcinoma; g, normal uterus; h, uterine endometrial adenocarcinoma. a to e and g, positive; f and h, negative. B, immunohistochemical staining for Annexin XI in two representative pairs of surgical specimens derived from primary and matched recurrent ovarian cancers of the same patient. All two paired cases showed decreased expression of Annexin XI in recurrent tumors (b and d) compared with their corresponding primary tumors (a and c). C, Kaplan-Meier curves of disease-free interval for patients with low Annexin XI-expressing (solid line; ≤2) and high Annexin XI-expressing (dashed line; >2) tumors (P = 0.03).

 
In addition, arrays containing various human malignancies were examined to determine whether expression of Annexin XI was altered in cancer tissues compared with the normal tissue from which they arose. The decreased expression of Annexin XI was observed in some of the most common human malignancies as compared with their cognate normal tissues. These tumor types included pancreas (12 of 18), kidney (6 of 20), stomach (13 of 18), colon (10 of 20), lung (7 of 15), bladder (11 of 18), prostate (2 of 10), uterus (9 of 19), breast (7 of 24), and ovary (11 of 31; Fig. 2A, e-h). No staining was detectable in cancer tissues of cerebrum (0 of 18), testis (0 of 18), fibrous tissues (0 of 20), and head and neck (0 of 17). Expression was only detected in a low fraction of cancer tissues of liver (3 of 19), thyroid (4 of 19), lymph node (4 of 18), and skin (2 of 14).

Decreased expression of Annexin XI correlates with in vitro cisplatin resistance and tumor recurrence in ovarian cancer patients. Ovarian cancer has generally been treated with cisplatin-based chemotherapy and often recurs due to acquired cisplatin resistance. To clinically confirm the involvement of Annexin XI in acquired cisplatin resistance, we evaluated the expression of Annexin XI in 164 ovarian carcinoma tissues with immunohistochemical staining. As shown in Table 3 , the expression level of Annexin XI in first recurrent ovarian cancers (57 cases) was much lower than that in primary ovarian cancers (93 cases; P = 0.0004). Approximately, 65% of first recurrent ovarian cancers exhibited decreased Annexin XI expression levels compared with primary ovarian cancers. Among 20 pairs of surgical specimens derived from primary and matched recurrent ovarian cancers of the same patients, 14 (70%) paired cases showed reduced expression of Annexin XI in recurrent lesions, whereas only 2 paired cases showed a reverse alteration (Fig. 2B). A statistically significant decrease in Annexin XI immunointensity (0 and 1+) was found in recurrent tumors as compared with the primary tumors from the same patients (P = 0.02). Annexin XI immunostaining was observed in both cytoplasm and nucleus of cancerous epithelial cells contrasting with the adjacent immunonegative stroma cells (Fig. 2B). No significant Annexin XI immunoreactivity was detected in negative controls that were analyzed with primary antibody omitted and control IgG1 (data not shown). Among 40 patients with advanced-stage serous carcinomas who underwent primary debulking surgery followed by a standard chemotherapeutic regimen, patients with low Annexin XI-expressing tumors (29 cases; ≤2) exhibited earlier recurrence than those with high Annexin XI-expressing tumors (11 cases; >2; P = 0.03; Fig. 2C). The median disease-free interval with Annexin XI immunointensity of ≤2 was 473 days, whereas when the intensity was >2, the interval was 755 days. Among 105 tumors for which the extreme drug resistance results were available for analysis, Annexin XI immunoreactivity inversely correlated with in vitro cisplatin resistance (P = 0.01; Table 4 ). The statistical significance in the association of Annexin XI immunoreactivity and in vitro cisplatin resistance existed even if only high-grade (2 or 3), advanced-stage (III or IV) serous carcinomas (67 tumors) were analyzed (P = 0.02; Table 4). More specifically, ~69% of ovarian carcinomas with extreme and intermediate cisplatin resistance exhibited low Annexin XI expression levels (0 and 1+), and 64.3% of high-grade, advanced-stage serous carcinomas with extreme and intermediate cisplatin resistance also showed low Annexin XI expression levels (0 and 1+). However, expression of Annexin XI did not correlate with the status of in vitro drug resistance for carboplatin (P = 0.15), paclitaxel (P = 0.57), and Taxotere (P = 0.78). There was no significant association of Annexin XI staining either in primary tumors or in recurrent tumors with either overall survival or any of the other clinical variables including stage, grade, or histologic subtype.


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  		Table 3. Decreased Annexin XI immunoreactivity correlates with tumor recurrence in ovarian cancer patients (P = 0.0004)

 

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  		Table 4. Decreased Annexin XI immunoreactivity correlates with in vitro cisplatin resistance in ovarian cancer patients

 

   Discussion
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 Abstract
 Materials and Methods
 Results
 Discussion
References
 
Cisplatin is the most common therapeutic agent used for chemotherapy in ovarian cancer. Acquisition of cisplatin resistance during chemotherapy is highly related to cancer mortality and remains a major clinical challenge. Cisplatin is a cytotoxic compound that causes apoptosis via DNA damage by formation of interstrand or intrastrand adducts. The response to cisplatin is a complex and multifactorial process that leads to the activation of several pathways organized in a large network and transmitting proapoptotic or antiapoptotic signals (24). The major mechanisms of resistance that have been identified thus far involve reduced drug uptake, increased drug efflux, increased repair of platinum-DNA adducts, increased tolerance of DNA damage, and increased levels of intracellular thiols such as glutathione and metallothionein (5–9, 25). Although many putative cisplatin resistance mechanisms have been identified and characterized in vitro, their relevance to clinical resistance has been difficult to prove. The mechanisms underlying clinical cisplatin resistance remain poorly understood because no single overarching mechanism predominates even within histologic subtypes of tumor. Application of new global approaches such as cDNA microarray and proteomics offers the promise of a more through understanding of the mechanisms that mediate cisplatin resistance and the possibility of developing strategies for individualizing therapy.

In this study, we used antibody microarrays to identify proteins consistently differentially expressed across multiple pairs of isogenic cisplatin-sensitive and cisplatin-resistant human ovarian cancer cell lines. The down-regulation of Annexin XI in cisplatin-resistant cells was subsequently validated by immunoblotting in all of three pairs of ovarian cancer cell lines. Decreased expression of some epigenetically controlled genes was reported to be typical for cisplatin-resistant ovarian cancer cell lines (25). However, very few mechanistic proteomic studies have been applied to describe chemoresistance emergence in ovarian cancers. We report here the identification of Annexin XI and its association with cisplatin-resistant phenotype by comparing multiple isogenic pairs of cisplatin-sensitive and cisplatin-resistant human ovarian cancer cell lines. A novel experimental setup and data analysis algorithm was used in the study to achieve a high confidence that the identified proteins are really markers of cisplatin resistance. The experimental results suggested that decreased expression of Annexin XI is characteristic for cisplatin-resistant ovarian cancer cells.

Annexin XI is a member of the Annexin superfamily of structurally related Ca2+-dependent, phospholipid-binding proteins. Despite their structural similarities, Annexins have diverse functions including cell division, apoptosis, Ca2+ signaling, growth regulation, and secretory function involving both exocytotic and endocytotic pathways (19–22). Annexin XI contains a conserved structural element, four tandem Annexin repeats, in which the Ca2+-binding sites are located; a unique NH2-terminal domain rich in glycine, proline, and tyrosine residues involved in binding to calcyclin (S100A6) and the apoptosis-linked protein ALG2 (26, 27). Annexin XI may have a role in cellular DNA synthesis and cell proliferation as well as in membrane trafficking events such as exocytosis (28–32). Previously, Annexin IV has been found less expressed in the cisplatin-resistant cell lines (IGROV1-R10 and IGROV1/CP) in comparison with the sensitive parental ovarian cancer cell line (IGROV1; refs. 23, 24). Several Annexin family members (Annexins I, II, IV, VII, and XI) were down-regulated in hormone-refractory prostate cancer (33). However, Annexin IV was found to be overexpressed in a paclitaxel-resistant lung cancer cell line (34). Annexin I has been linked to resistance to doxorubicin, melphalan, and etoposide in MCF-7 cells via a mechanism that is independent of multidrug resistance protein 1 and P-glycoprotein 1, but is linked to enhanced vesicle trafficking (35). Thus, Annexins seem to play a role in drug resistance of various cancers. Different drugs may have different effects on the expression of certain proteins. The identification of Annexin XI associated with cisplatin resistance in vitro in this study warrants further investigation of its clinical relevance in human ovarian cancer patients.

The immunohistochemical analysis showed that Annexin XI was expressed in majority of human normal organs including pancreas, kidney, stomach, colon, lung, bladder, prostate, uterus, placenta, breast, and ovary. This analysis further underscored that Annexin XI was a ubiquitously expressed protein in a variety of tissues and cell types of eukaryotes (28, 31), which was in agreement with its multifunctional nature (28–32). Although diverse functions have been ascribed to Annexins, there is no consensus about the role played by this family as whole (19). Very little data existed on the implication of Annexin XI in cancer; however, taking the functions of Annexins into consideration, certain modifications in the expression of these proteins could have important consequences on cancer cell behavior. In this study, the decreased expression of Annexin XI was observed in some of the most common human malignancies, including pancreas, kidney, stomach, colon, lung, bladder, prostate, uterus, breast, and ovary, compared with their cognate normal tissues. These results indicated that the regulation of this protein was altered at some point during tumorigenesis. Paradoxically, Annexin XI was shown to be increased in malignant tissue when compared with the adjacent normal tissue in a patient with primary breast cancer using antibody microarrays (36). Several autoantigens including Annexin XI-A could significantly discriminate between breast cancer and noncancer control sera and differentiate patients with ductal carcinoma in situ from those with invasive ductal carcinoma of the breast (37). More detailed comparison of Annexin XI expression in cognate normal and malignant tissues is needed.

Thus far, most Annexins are believed to be localized underneath the plasma membrane or to be associated with the cytoskeleton (29). In contrast, the immunofluorescence study revealed both a cytoplasmic and a nuclear staining pattern for the 56K autoantigen (Annexin XI; ref. 29). Annexin XI was found to be either predominantly cytoplasmic in some cell types or nuclear in others. The nuclear localization of Annexin XI has been shown to be cell type specific and developmentally dependent (28). Consistent with these observations, Annexin XI immunostaining was observed in both cytoplasm and nucleus of ovarian cancer cells in this study. Moreover, there was an inverse correlation between Annexin XI expression and recurrence status in this panel of ovarian cancer patients, which has important biological and clinical relevance. The expression level of Annexin XI in first recurrent ovarian cancers was significantly lower than that in primary ovarian cancers. This indicated either that platinum/paclitaxel-based chemotherapy altered the Annexin XI gene regulatory controls or that chemotherapy destroyed the population for cells that had high levels of this protein. Decreased expression of Annexin XI may partially contribute to tumor recurrence. Annexin XI immunoreactivity inversely correlated with in vitro drug resistance for cisplatin, which further suggested that Annexin XI expression is important for acquired cisplatin resistance in human ovarian cancers. It is also important to note that it remains possible that the alteration of Annexin XI expression observed in this study was caused by carboplatin or paclitaxel rather than cisplatin that the patient received. Whether Annexin XI is directly involved in the clinical resistance to cisplatin requires further confirmation. Furthermore, increased Annexin XI immunoreactivity in ovarian cancers seemed to prolong the disease-free interval of patients. The low Annexin XI immunoreactivity in primary tumors is highly predictive of a shorter disease-free interval independent of other clinical variables including stage, grade, or histologic subtype. The current study validated only one of the candidate biomarkers generated from the discovery study. It is possible that additional validation studies of other candidates, such as MUPP1, could prove to be complementary to Annexin 11 when they are used in combination and have a better overall predictive performance. Annexin XI expression may potentially be used alone or in combination with other markers as a predictive marker of chemoresistance for platinum-based chemotherapy and to identify ovarian cancer patients who are likely to develop early recurrence.

In conclusion, this study showed that Annexin XI is associated with acquired cisplatin resistance in ovarian cancer. The decreased expression of Annexin XI is characteristic for cisplatin-resistant cancer cells and may partially contribute to tumor recurrence. Annexin XI expression may potentially be used as a predictive marker of chemoresistance for platinum-based chemotherapy and to identify ovarian cancer patients who are likely to develop early recurrence. Modulation of Annexin XI expression may represent a novel therapeutic strategy in human ovarian cancers.


   Footnotes
 
Grant support: Department of Defense IDEA grant DAMD17-OC03-IDEA, National Cancer Institute Early Detection Research Network grant CA115102-01, and National Cancer Institute grant 1P50 CA83639, The University of Texas M. D. Anderson Cancer Center Specialized Programs of Research Excellence (SPORE) in Ovarian Cancer.

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

3 http://bioinfo2.clontech.com/abinfo/array-layout-action.do?catalog_no=631790_6070032 Back

Received 3/ 8/07; revised 6/15/07; accepted 8/22/07.


   References
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