Background
Ovarian cancer ranks third among malignant tumors of the female reproductive system; however, it is the leading cause of cancer-related mortality, which seriously threatens women’s lives and health [
1,
2]. Metastasis and invasion of early-stage ovarian cancer is a major factor responsible for its high mortality and poor prognosis. Therefore, elucidating the mechanisms underlying the development and progression of ovarian cancer at a molecular level is important to facilitate the early diagnosis and treatment of ovarian cancer and to improve the prognosis of patients with this disease.
Human epididymis protein 4 (HE4), also known as whey acidic protein, was first shown to be highly expressed in ovarian cancer in 1999 [
3], and it was identified as a serum marker for ovarian cancer in 2003 [
4]. HE4 is highly expressed in epithelial ovarian cancer, whereas it is present at low levels in normal tissues, tumor-adjacent tissues and benign tumors [
3,
5]. HE4 has higher sensitivity, specificity, positive likelihood ratio and negative likelihood ratio than CA125 for the diagnosis of ovarian cancer [
6]. As a secreted glycoprotein [
7], HE4 has a significantly lower molecular weight than CA125, which has received wide attention. However, little is known about the function of HE4, specifically the role of HE4 in the malignant biological behavior of ovarian cancer. Recent studies showed that HE4 mainly affects the invasive and metastatic ability of ovarian cancer cells [
8,
9]; however, the underlying mechanism remains unclear. Whether HE4 acts alone or through interaction with a receptor on the cell membrane to affect the malignant biological behavior of ovarian cancer cells remains unknown.
In the present study, we showed that annexin II (ANXA2), a specific binding partner of HE4, is expressed in ovarian cancer cells, and the interaction between HE4 and ANXA2 promotes the invasion and metastasis of ovarian cancer cells via the MAPK and FOCAL signaling pathways.
Discussion
Serum HE4 detection is widely used for the diagnosis and monitoring of epithelial ovarian cancer. However, HE4 is not considered a target for the clinical treatment of ovarian cancer largely because its role in the development and progression of ovarian cancer is unclear. In the present study, we identified ANXA2 as an HE4 interacting protein using MALDI-TOF-MS, and their binding interaction was validated using co-immunoprecipitation and confocal laser scanning microscopy. HE4 and ANXA2 interaction was then validated in three ovarian cancer cell lines, which suggested that such an interaction is present in ovarian cancer tissues. In addition, pull-down assays revealed that the HE4 and ANXA2 binding site is located after the 26th amino acid at the N terminus.
Recent studies showed that HE4 promotes ovarian cancer cell invasion and metastasis [
8,
9]. Our previous study validated this finding (unpublished data); however, the underlying mechanisms remained unclear. To further explore the mechanisms underlying the effect of HE4 on the invasion and metastasis of ovarian cancer cells after its secretion to the extracellular medium, EGFP-transfected ES-2 cells were dynamically observed for 5 h. The results showed that the HE4 protein was not only expressed in the cytoplasm and peri-nuclear regions of ES-2 cells, but also in the nucleus. Furthermore, HE4 secreted to the extracellular region bound to the membrane of the ES-2 cells themselves and to that of neighboring cells. The binding of HE4 to cell membrane proteins may play a decisive role in the malignant biological behaviors of ovarian cancer cells, such as invasion and metastasis.
ANXA2 is a calcium-dependent phospholipid binding protein that is mainly located on the cell membrane. ANXA2 is an S100 protein family member and a fibrinolytic receptor for the S100A4 protein [
11]; annexin II is a membrane protein. The ANXA2 and S100A4 interaction can promote tissue-type plasminogen activator (t-PA)-dependent plasmin generation and activation of its downstream matrix metalloproteinases (MMPs), including that of matrix metalloproteinase-2 (MMP-2). This results in extracellular matrix (ECM) remodeling and neovascularization, which promote the invasion and metastasis of tumor cells [
12‐
14]. Changes in the expression and spatial distribution of ANXA2 are closely associated with the invasion and metastasis of multiple tumors, and the interaction between ANXA2 and molecules involved in tumor invasion and metastasis may promote these malignant behaviors. The present study is the first to demonstrate the structural relationship between HE4 and ANXA2, which led to the hypothesis that HE4 and ANXA2 binding promotes ovarian cancer cell invasion and metastasis. To test this hypothesis, we generated ovarian cancer cell lines with high and low HE4 expression and showed that the up- or downregulation of HE4 was accompanied by parallel changes in ANXA2 expression in the treated cell lines. Interference with
HE4 significantly inhibited HE4 and ANXA2 co-localization on the cell membrane, as shown by confocal laser scanning microscopy. In addition, the reduction in the invasive and metastatic abilities of cancer cells induced by ANXA2 downregulation were not reversed by HE4 active protein supplementation, whereas upregulation of ANXA2 expression restored invasion and migration and this effect was enhanced by exogenous HE4 active protein. These results indicated that ANXA2 may act cooperatively with HE4 in promoting the invasion and migration of ovarian cancer cells. Immunohistochemical analysis of clinical specimens showed that HE4 and ANXA2 protein expression levels were higher in malignant and borderline epithelial ovarian tissues than in benign epithelial ovarian tumor tissues. In addition, the two proteins were expressed at higher levels in ovarian cancer tissues with lymph node metastasis than in those without (all P < 0.05). The histological results confirmed the association of HE4 and ANXA2 expression with the degree of malignancy of ovarian cancer.
Recent studies showed that upregulation of ANXA2 expression activates the MAPK signaling pathway and promotes the malignant biological behaviors of tumor cells, such as proliferation [
15], invasion and metastasis [
16,
17]. Serial analysis of gene expression showed that upregulation of ANXA2 activates
RPS6KA1, a downstream component of the MAPK signaling pathway, thereby affecting the development and progression of gallbladder cancer [
18]. In addition, HE4 was found to promote the invasion and metastasis of ovarian cancer cells via the EGFR/MAPK pathway [
8]. Our findings indicated that MAP kinase interacting serine/threonine kinase 2 (MKNK2) and laminin beta 2 (LAMB2) gene expression levels were downregulated in response to
HE4 interference in ovarian cancer cells, whereas exogenous HE4 protein supplementation reversed this effect. Our results suggest that HE4 and ANXA2 binding activates the MAPK and FOCAL adhesion signaling pathways, thereby promoting the invasion and metastasis of ovarian cancer cells. Our results indicate that annexin II may help HE4 translocate into the nucleus, where it functions as a transcription factor promoting the expression of MAPK or FOCAL signaling molecules.
Tumor invasion and metastasis are complex pathophysiological processes that include not only interactions between tumor cells and between tumor cells and host cells, but also a complex regulatory network involving multiple bioactive molecules. A recent study showed that binding of HE4 to MMP2 and MMP9 in renal cells promotes renal fibrosis [
19]. ANXA2 was shown to promote the invasion and metastasis of ovarian cancer cells through the activation of MMP2 [
12‐
14]. These findings together with those of our previous studies suggest that HE4 is secreted to the extracellular medium, where it binds to ANXA2 on the cell membrane, activating downstream signaling molecules and inducing changes n the cell nucleus. Furthermore, the HE4 and ANXA2 complex may promote the invasion and metastasis of ovarian cancer cells by activating MMPs and promoting ECM remodeling. S100A4 and ANXA2 binding promotes invasion and signal transduction in tumor cells [
11]. Further studies are necessary to investigate the role of S100A4 in the interaction between HE4 and ANXA2 in ovarian cancer cells. Elucidation of the biological functions of HE4 will reveal the mechanisms underlying the role of HE4 in the development, invasion and metastasis of ovarian cancer and may lead to the design of therapeutic strategies targeting HE4 and ANXA2 for the treatment of ovarian cancer.
Methods
Construction of expression vectors
Reverse transcription-PCR products produced from human cDNA and corresponding to full-length human HE4 and annexin II were cloned in pGEX-4 T or pGEX-6 T vectors (Amersham Biosciences). N-terminal truncated forms of annexin II with its first 15 or 26 amino acids deleted, A2-del15 and A2-del26, respectively, for GST pull-down assays. Glutathione S-transferase fusion proteins were purified using glutathione-Sepha-rose 4 beads and cleaved with thrombin or PreScission protease to remove glutathione S-transferase tag according to the manufacturer’s instructions (Amersham Biosciences). An HE4 expression construct was generated by subcloning PCR-amplified full-length human HE4 cDNA into the pEGFP-N1 or pCMV6 plasmid. The following primers are used: P1:5’- TCC GCT CGA GAT GCC TGC TTG TCG CCT AG -3’和P2:5’- ATG GGG TAC CGT GAA ATT GGG AGT GAC ACA GG -3’. Two shRNA expression vectors for human HE4 were constructed using the vector pSilence. The mRNA target sequences chosen for designing HE4-shRNA are GTC CTG TGT CAC TCC CAA T for HE4-shRNA1 and GAT GAA ATG CTG CCG CAA T for HE4-shRNA2. Two shRNA expression vectors for human ANXA2 were constructed using the vector pSilence. The mRNA target sequences chosen for designing ANXA2-shRNA are GTA CTA TTA TAT CCA GCA A for ANXA2-shRNA1 and AGG AAA TTA ACA GAG TCT A for ANXA2-shRNA2.
Cell culture and transfection
OVCAR-3,SKOV-3,ES-2 and CaoV-3 ovarian cancer cell lines were purchased from American Type Culture Collection and propagated in McCoy’s 5A modified medium with 10% fetal bovine serum. Transfection was carried out using liposomes with a vector transfection kit according to the instructions. Stable cell lines expressing HE4 and ANXA2 shRNAs were selected for 14 days with 800 ug/ml G418 (Invitrogen).
Immunoprecipitation, silver staining, and protein identification by mass spectrometry
OVCAR-3 cell was immunoprecipitated using an anti-HE4 antibody (Santa Cruz, goat, CA) and combined with 30 ul of protein A/G PLUS agarose (Santa Cruz) by rotating for 1 h at 4°C. The eluents were loaded onto SDS-PAGE gel and Coomassie brilliant blue-stained. The expressed bands were excised and processed for in-gel trypsin digestion and subjected to MALDI-TOF-MS analysis. Anti-HE4 antibody was replaced by goat IgG (Bioss, China) for negative control. The peptide and proteins were identified from the MS/MS spectra using the MASCOT algorithm (Matrix Science, Boston, MA). Peptide mass fingerprinting was carried out using the MASCOT search engine from GPS Explorer software (Applied Biosystems, Foster City, CA). Mass spectra used for manual denovo sequencing were annotated with the Data Explorer soft-ware (Applied Biosystems).
Co-immunoprecipitation and Western Blot
Ice-cold RIPA buffer (1 ml) was added to ovarian cancer cells, followed by incubation at 4°C for 30 min. After centrifugation at 15,000 ×
g for 30 min at 4°C, supernatant fractions were collected and treated with anti-HE4 antibody (10 μl) (Santa Cruz, goat polyclonal) or anti-annexin II (Proteintech, mouse monoclonal) for 3 h at 4°C. Protein A/G PLUS-Agarose (20 μl; Santa Cruz)was added, followed by incubation on a rocker platform overnight at 4°C. The procedure was followed as described previously [
20]. The negative control contained only 10 μl HE4 or ANXA2 antibody without protein. Immunoprecipitates were subsequently subjected to 12% SDS gel electrophoresis and analyzed via Western blot using HE4 monoclonal (Abcam, Rabbit) and annexin II monoclonal (Abcam, Mouse) antibodies. Proteins were visualized using ECL reagent (Amersham ECL Prime detection). Experiments were repeated three times.
Cytoplasmic and membrane proteins extraction
Cytoplasmic and membrane proteins were extracted according to the instructions of the membrane and cytoplasmic protein extraction kit (Beyotime, Haimen, China). Membrane protein extraction reagent A (1 ml) was added to 5 × 107 cells, followed by incubation at 4°C for 15 min. After centrifugation at 700 g for 10 min at 4°C, supernatant fractions were collected carefully and kept as cytoplasmic proteins. The precipitate was centrifugated at 14,000 × g for 30 min at 4°C. Then, membrane protein extraction reagent B (300 ul) was added after the supernatant fractions were discarded, and the mix was vortexed violently for 5 sec followed by incubation at 4°C for 15 min. These steps were repeated twice. After centrifugation at 14,000 × g for 5 min at 4°C, supernatant fractions were collected carefully and kept as membrane proteins.
Pull-down assay
Bacterial lysate expressing His-tagged ANXA2 protein was purified using HisTrap Kit (Amersham Biosciences). Purified His-tagged ANXA2 was incubated with GST-HE4 immobilized on 100ul of glutathione-Sepharose beads (GE Healthcare). Beads were extensively washed with Buffer A (20 m M Tris–HCl (pH 8.0), 1 mM EDTA, 1 mM dithiothreitol, 150 mM NaCl, 1% Triton X-100) containing 1ul protease inhibitor mixture. The bound proteins were eluted by boiling in the SDS sample buffer for 10 min and immunoblotted with an anti-His-tagged antibody (GeneTex).
Sandwich ELISA
Ninety- six-well polystyrene microplates were coated with a capture antibody against HE4 (Santa Cruz, goat polyclonal) at 5 μg/ml in coating buffer at 4°C for 16 h. After blocking with 5% BSA, 100 μl of the cell supernatants were added to the wells and incubated at room temperature for 2 h followed by peroxidase-labeled anti-goat IgG antibody. Color reaction was developed with o-phenylenediamine dihydrochloride solution at room temperature for 20 min. The reaction was stopped with 2.5 M sulfuric acid. Negative controls were performed with 1% BSA instead of the mAbs. The optical density of each well was determined within 30 min using a microplate reader at 450 nm [
21].
Immunohistochemistry and immunocytochemistry
Histological section of each group of ovarian tissue was 5 μm. Each tissue had two serial sections. Expression patterns of HE4 and ANXA2 in ovarian carcinoma tissues were analyzed via immunohistochemical streptavidin-peroxidase staining. Positive and negative immunohistochemistry controls were routinely employed. Normal epididymis tissue served as a positive control for HE4, while breast cancer tissue was used as the positive control for ANXA2 antigen. The negative control was incubated with rabbit IgG (Bioss, China) instead of primary antibody. The working concentrations of primary antibodies against HE4 and ANXA2 used were 1:40 (Abcam, Rabbit polyclonal to HE4) and 1:1200 (Abcam, Rabbit polyclonal to ANXA2), respectively. The empirical procedure was performed based on the manufacturer's instructions.
Cells at exponential phase of growth were digested by 0.25% trypsin and cultured in medium containing 10% FBS to prepare single-cell suspension. Cells were washed twice with cold PBS when growing in a single layer, and fixed with 4% para- formaldehyde for 30 min. The expression of annexin II and HE4 on cells were detected according to the SABC kit instructions. The working concentrations of primary antibodies against HE4 and ANXA2 used were 1:300 (Abcam) and 1:1000 (Abcam), respectively. The primary antibody was replaced by rabbit IgG for negative control. The average optical densities were measured under a microscope with image processing, being presented as the means ± standard deviation for three separate experiments.
Double-labeling immunofluorescence method
Cells at exponential phase of growth were digested by 0.25% trypsin and cultured in medium containing 10% FBS to prepare single-cell suspension. Cells were washed twice with cold PBS when growing in a single layer, and fixed with 4% para- formaldehyde for 30 min. The cells were simultaneously incubated with primary antibodies against HE4 (1:100, Abcam, Rabbit) and annexin II (1:50, Proteintech, Mouse). The primary antibody was replaced by rabbit or mouse IgG for negative control. The working concentrations of fluorescein isothiocyanate and TRITC were 1:100. Nuclei were counterstained with DAPI. The empirical procedure was performed according to the manufacturer's instructions.
Scratch test and the transwell assay
Scratch test: Cells during the log phase were selected and single cell suspensions were prepared. Cells on a 6 well plate were cultured until 90% density. And then the plate was scratched with a 200ul pipette tip. Cells were cultured in medium without serum. After 12 h, the width of the scarification were observed under microscope. Transwell assay: The Matrigel were melted and put at 4°C refrigerator overnight the day before this experiment. The pipette tip was pre-cooled in ice-cold for 0.5 h during experiment, and the ECM gel was diluted by 1:8 with serum free medium, Matrigel 100 ul was added into the upper chambers, the whole process was performed on ice. Then they were placed in an incubator at 37°C for 5 h. 105/mL cells in logarithmic growth phase was added in each well for 200 ul, 500 ul medium supplemented with 10% fetal bovine serum were added in lower chamber. After culturing for 24 h, nutrient solution was abandoned and a cotton swab was used to gently wipe out the upper layer of transwell. Membrane of transwell was fixed with methanol for 20 min, washed with PBS 3 times, then staining with 0.1% crystal violet for 20 min after airing. The invasive cell numbers of 5 fields (upper and lower, left and right, middle) were counted under microscope, the mean value was obtained and the statistical analysis was made. The cells of each group were treated in triplicate and experiments were repeated three times.
Antibody blocking tests
Cells during the log phase were selected and single cell suspensions were prepared. Anxa2 mAb (10 μg/ml) was added to the adherent cells. Mouse IgG isotype control and PBS blank control groups were generated. The cells were incubated at 37°C for 30 min. The experiments were repeated three times and the average value was calculated. In the Transwell assay, Anxa2 mAb (10 μg/ml) was added into the upper chamber while paving the cells. The cells of each group were treated in triplicate and experiments were repeated three times.
Transgenic mice and tail-vein injections
Female BALB/c nu/nu mice (4–6-weeks-old; Shanghai Institute of Material Medicine, Chinese Academy of Science) were raised in specific pathogen-free conditions. All experimental protocols were approved by the Committee for the Care and Use of Laboratory Animals of Shengjing Hospital Affiliated with China Medical University., the permit numbers is 2014PS163K.
Tail-vein injection experiments and peritoneal metastasis assays were performed on
nu/nu mice obtained as described elsewhere [
22]. The 30 mice were divided into three groups randomly, namely ANXA2 high expression, ANXA2 low expression and mock groups. Cells at the exponential phase of growth were digested by 0.25% trypsin and resuspended in PBS. A volume of 200 μl (1 × 10
6) was injected into the tail-vein of
nu/nu mice and the mice were observed for 24 days. Cells (5 × 10
6 cells/200 μl PBS) were injected intraperitoneally for peritoneal metastatic formation and the mice were observed for 25 days. The mice were then killed humanely and an autopsy was performed and the lungs and the peritoneal were examined for tumours separately. Then, tissues were dehydrated, processed, and embedded in paraffin wax. Serial sections 5-μm thick were prepared from each block, stained with haematoxylin and eosin (H&E) and analyzed by immunohistochemistry (HE4 antibody, 1:3000; ANXA2, 1:4000).
Time lapse observation
Cells during the log phase were selected and single cell suspensions were prepared. Cells on a Confocal dish were cultured until 60% density. And then observe the cells using confocal microscope (Nikon A1) in 5%CO2, 37°C for 5 h. Continuous shooting at every 5 minutes. The experiments were repeated three times (see supplementary material).
Ethics statement
Samples were fully encoded to protect patient confidentially. The study and its protocols were approved by the Research Ethics committees of Shengjing Hospital Affiliated with China Medical University. IACUC permit number is 2013PS23K. Because all the samples used in the study were discarded, the informed consents were not needed. And the ethics committees approved this.
Patients and tissue samples
Selected paraffin samples (107 in total) were obtained from the operations performed from 2004 to 2012 in the Department of Gynecology and Obstetrics of our hospital. All tissue sections were examined by specialists to obtain a final diagnosis. Normal ovarian samples were obtained from tissue excised in cervical cancer operations. Among the benign ovarian tumors, six cases were mucinous cystadenoma, nine cases were serous cystadenoma. There were 27 cases of borderline ovarian tumors (including 13 mucinous and 14 serous cystadenomas). The mean age of these patients was 46.97 years (16–81 years). The age range of the ovarian cancer group was 16 to 73 years; median age was 53 years. The age range of the borderline ovarian tumor group was 22 to 77 years; median age was 36 years. The age ranges of the benign ovarian tumor and normal tissue groups were 22 to 81 years and 37 to 59 years, respectively; median ages were 44 and 50.5 years, respectively. Comparing these groups, there is no statistical significance (P > 0.05). Specific histological types and pathological grades are presented in Tables
1 and
2.
All cases were primary, and the information was complete. All the patients had received a Gastroscopy or colposcopy to exclude other primaries. Patients were not subjected to chemotherapy prior to the operation.
Assessment standard
Immunohistochemistry
We consider a positive result if there are buffy granules in the cell membrane and cytoplasm. According to the chromatosis intensity, no pigmentation, light yellow, buffy, and brown are scored 0, 1, 2, and 3, respectively. We choose 5 high-power fields in series from each slice, then score them and take the mean percentage of the chromatosis cells: chromatosis cells that account less than 5% are 0, 5% to 25%; 1, 26% to 50%; 2, 51% to 75%; 3, and greater than 75%; 4, Multiply these 2 numbers; 0 to 2 is considered (−); 3 to 4, (+); 5 to 8, (++); and 9 to 12, (+++). Two observers read the sections to control error. At the same time, we use the NIS-Elements BR 2.10 picture analysis software of the Japanese Nikon Company (Tokyo, Japan) to measure the mean optical density (MOD).
Statistical analysis
SPSS version 17.0 (SPSS Inc, Chicago, IL) software was used for statistical analysis. χ
2
analysis, variance analysis, and t-test were employed. P < 0.05 was considered statistically significant.
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Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
ZHY and LB carried out the molecular genetic studies and drafted the manuscript. HZH and TMZ carried out the immunoassays. LDW and ZLC participated in the design of the study and performed the statistical analysis. GJ conceived of the study, and participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript.