Background
Among women, ovarian cancer is one of the most common gynecological cancers worldwide. With the highest mortality rate of all gynecologic cancers, ovarian cancer is very problematic to treat. Even after optimal treatment, more than half of patients suffer recurrence and eventually die [
1].
Tumor progression is generally associated with extensive tissue remodeling to provide a proper environment for tumor growth, angiogenesis, invasion, and eventual metastasis of cancer cells. It is known that proteases are key agents in tumor progression, and that naturally expressed protease inhibitors have the ability to counteract tumor progression and metastasis [
2‐
4]. However, expression of serine protease inhibitors (SPIs) in tumors is often associated with poor prognosis in cancer patients. Moreover, there is growing evidence that SPIs may even promote cancer cell malignancy which, if confirmed, could make them useful biomarkers of malignancy [
5].
A recent study has identified
WFCD2 as a new member of the group of serine protease inhibitors belonging to the WAP family. While prior research indicated a direct linkage between
WFCD2 expression and cell proliferation [
6,
7], its physiological and pathological mechanisms in tumorigenesis and metastasis have not been clearly elucidated.
Human
WFCD2 gene located on chromosome 20q12–13.1 locus, which encode a serial of proteins with a WAP-type four disulphide core (WFDC) domain [
8,
9]. More and more evidence suggests that overexpression of WAP-type proteins closely related to tumor metastasis, especial
SLPI and
P13 (encode antileukoproteinase 1 and elafin respectively). Both
SLPI and
P13 are co-expressed with
WFCD2 and have been identified as a promoter in cancer development in various carcinomas [
10,
11]. Expression of
SLPI is positively correlated with increased expression of the cell cycle progression factor Cyclin D1 [
12,
13], and its causal role in the promotion of malignant behavior has also been demonstrated in lung carcinoma cells stably transfected with human
SLPI-expression constructs [
14]. Elafin (
P13) also has a role in counteracting environmental proteolytic conditions and repair-processes that are commonly associated with the inflammatory response, cancer progression, and invasion of various tumor cells [
3,
15].
In view of the above information, the WAP proteins, had been considered as being associated with high-risk, metastatic, or aggressive cancer originating from various organs [
9,
16]. We speculated that
WFCD2 might also play some role in tumor progression in ovarian cancer.
Our previous study indicated that knockdown of
WFCD2 induced the up-regulation of Fasl and down-regulation of Cyclin D1, as well as activating Caspase 3 and Ki67 [
6]. These results indicate that
WFCD2 plays very important roles in tumor formation and proliferation. In the presented study, we analyze the expression of
WFCD2 in ovarian cancer cell line HO8910 and aggressively malignant line HO8910-PM. A cell model of
WFCD2 gene down-regulation was constructed and used to analyze the function of
WFCD2 in tumor metastasis and tumorigenesis in vitro and in vivo
.
Methods
Ethic statement
Ovarian tumors were obtained from a cohort of patients treated at Nanfang Hospital, affiliated with Southern medical University, China, between 2011 and 2014. All research involving human ovarian cancer tissues have been approved by Nanfang hospital ethics committee and written consent was obtained from all participants. The 6- to 8-week-old female BALB/c–nu mice used in these experiments were provided by the experimental animal center of the Southern Medical University (Guangzhou, China). All mouse studies were approved by the Animal Ethics Committee of the Southern Medical University (Permit Number 20060015). All work was undertaken and that it conforms to the provisions of the
declaration of Helsinki (as revised in Fortaleza, Brazil, October 2013).
Patients and tissue samples
The median age of the patients was 50.8 years. All patients were diagnosed by pathological analyses based on the International Union Against Cancer (UICC) tumor node metastasis (TNM) stage system. 100 tissue samples (Table
1) from normal ovarian, primary tumors and matched adjacent non-neoplastic ovarian tissues were collected and prepared for Anti-
WFCD2 polyclonal antibody (Abcam, Cambridge, MA, USA) was used as primary antibody. The staining intensity (0, no staining; 1, weak staining; 2,moderate staining; and 3, intense staining) and the proportion of stained cells (0, no staining; 1, <10% staining; 2, between 11 and 33% staining; 3, between 34 and 66% staining; and 4, >67% staining) were semiquantitatively determined. The intensity and the percentage of positive cell scores were multiplied (0–12) and classified into three groups: weak (0–4), moderate (5–8) and strong (9–12). All slides were scored by two observers blinded to the pathology and the clinical features.
Table 1
Distribution by tumor characteristics for ovarian cancer patients
Total |
Age(years) | | |
≤ 50 | 38 | 38 |
> 50 | 62 | 62 |
FIGO stage |
Stage I | 26 | 28.57 |
Stage II | 21 | 23.08 |
Stage III | 31 | 34.07 |
Stage IV | 12 | 13.19 |
Grade(Epithelial, n = 91) |
G1 | 29 | 31.87 |
G2 | 46 | 50.55 |
G3 | 16 | 17.58 |
Histological type |
Serous cystadenocarcinoma | 46 | 50.55 |
Mucinous cystadenocarcinoma | 22 | 24.18 |
Endometrioid tumor | 14 | 15.38 |
Clear cell cacinoma | 9 | 9.89 |
Transcoelomic Metastasis |
No | 65 | 71.42 |
Yes | 26 | 28.57 |
Lymph node metastasis |
No | 74 | 81.31 |
Yes | 17 | 18.69 |
Cell lines and reagents
Human ovarian cancer cell lines SKOV3, HO8910 and HO8910-PM were purchased from the cell bank of the Chinese Academy of Sciences (Shanghai, China). The cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) media supplemented with 10% fetal calf serum (FBS) in an atmosphere of 5% CO2 at 37 °C. Restriction enzymes from TAKARA (TaKaRa Bio, Inc., Shiga, Japan); Transwell system from CostarCorning (Corning, NY, USA).; Puromycin and Trizol reagent from Invitrogen (Life Technologies, Carlsbad, CA, USA); cell culture media (antibiotic, serum and glutamine) from GIBCO (Life Technologies, Carlsbad, CA, USA). All other molecular reagents and solvents were purchased from SIGMA Corp (St. Louis, MO, USA).
Gene knockdown
WFCD2 knockdown was conducted in low-passage (<20) ovarian cancer cells. The shRNA oligo sequences were designed to against the human WFCD2 gene (Gene Bank Accession No. NM_0006103.3). The shRNA sequence against WFCD2(5′-GCTCTCTGCCCAATGATAAGG-3′) and a invalid RNAi sequence(5′-GTTCTCCGAACGTGTCACGT-3′) were chemically synthesized and constructed into the lentiviral by Shanghai Genepharma Co.Ltd. The WFCD2-specific shRNA lentivirus paticals was collected and transfected into the HO8910 and SKOV3 cell lines. For stable knockdown of WFCD2, the transfected HO8910 and SKOV3 cell lines, named HO8910–209 and SKOV3–209 respectively, were selected by Puromycin. Puromycin-resistant colonies were picked and expanded separately.
RNA extraction and real-time RT-PCR
Total RNA was isolated with Trizol regents and reverse transcription was performed using the PrimeScript 1st Strand cDNA Synthesis Kit, according to the manufacturer’s instructions, cDNA samples (0.1 μg) were assayed in duplicate using the ABI Prism 7500 detection system (Life Technologies, Carlsbad, CA, USA). Using the SYBR Green PCR Master Mix (TaKaRa) following protocols. The relative quantization number was then calculated by subtracting the average CT from the corresponding average CT for β-actin.
Tumour migration assay
Transwell polycarbonate plates with 6.5 mm diameter tissue culture inserts containing a membrane with 8 μm pores were used for migration assay. Low passage (<20) cells were cultured in the medium without serum to synchronize most of them at G1/G0 and then suspended in serum-free DMEM and seeded (5 × 104 cells/well) into each insert. The condition medium with 10% FBS collected after 24 h culture was added to each outer well. The plates were then assembled and incubated for 8 h at 37 °C, 5%CO2. After a 8 h incubation, the plates were rinsed once in PBS, fixed in 70% alcohol for 10 min, and rinsed with 0.5% crystal violet. Cells adhering to the top surface of the tissue culture inserts were removed with a cotton tip applicator, while cells adhering to the bottom surface of the inserts were rinsed with 1% Triton-X 100 in PBS for 20 min. The membranes of the tissue culture inserts were viewed under amicroscope (10× magnification) and the number of cells in 4 random fields was determined.
Tumour invasion assay
For invasion assays, 5 × 104 cells (cell passage <10) were cultured in the medium without serum to synchronize most of them at G1/G0 and then plated in DMEM/1% FBS in a cell invasion chamber (Transwell Cell invasion assay kit, Corning) in a 24-well plate, which contained an 8 μm pore size polycarbonate membrane covered with a thin layer of collagen matrix. Invasive cells migrated through a membrane according to the gradient of FBS to the lower chamber, which contained DMEM/15% FBS. The invasive cells were stained with crystal violet, and the number of cells in 4 random 10× magnification fields was determined.
For the generation of intraperitoneal tumors, HO8910-NA cells and HO8910–209 cells were injected intraperitoneally (i.p.) into mice (n = 10). Each mouse received one injection of 3 × 106cells. Animals were monitored 3 times weekly for tumor formation. All injection-treated mice were fed for 10 weeks after injection. At the end of 10 weeks, all the mice were sacrificed and the abdominal region examined for tumor formation. Each tumor burden in the peritoneal cavity was weighed and collected and paraffin-preserved according to the usual protocols.
Western blot
Total protein was extracted by sonication in radio-immunoprecipitation assay (RIPA) buffer(50 mM Tris–HCl pH 7.5, 150 mM NaCl, 5 mM EDTA, 0.5% Nonidet P-40, 5 mM dithiothreitol, 10 mM NaF, protease inhibitor cocktail). 100 μg denaturedprotein was separated on an SDS-polyacrylamide gel and transferred to Hybond membrane (Amersham, Germany), which was then blocked overnight in 5% skimmed milk inTris-bufferedsaline with Tween 20 (TTBS, 10 mM Tris–HCl, 150 mM NaCl, 0.1% Tween 20). For immunoblotting, the membrane was incubated for 15 min with antibodies. The membrane was rinsed with TBST and incubated with anti-mouse, anti-rabbit or anti-goat IgG conjugated to horseradish peroxidase (DAKO, USA, 1:1000) for 15 min. All the incubations were performed in a microwave oven to allow intermittent irradiation. Bands were visualized with LAS4010 (GE Healthcare Life Science, USA) by ECL-Plus detection reagents (Santa Cruz, USA). Densitometric quantification of protein bands was performed with GAPDH as an internal control using Image J (NIH, USA).
Immunohistochemistry
Immunohistochemistry was done using a single-staining procedure. Anti-WFCD2, anti-Ecadherin,anti-Vimentin monoclonal antibody (Cell Signaling Technology), anti-CD44,anti-MMP2,anti-MMP9,and anti-ICAM-1 rabbit polyclonal antibody (Santa Cruz Biotechnology), were applied to the slides at a dilution of 1:1,00 ~ 1:150 in blocking buffer overnight at 4 °C. The slides were then washed and stained by the avidin-biotin method. The slides were lightly counter stained with hematoxylin. Tumor cells were considered positive for the antigen if there was brown color staining. The intensity was scored as negative (0), weak (1), medium (2), and strong (3),and the proportion of staining was scored as 1 (≤10%), 2 (11–50%), 3 (51–75%), and 4 (>75%). An overall expression score was calculated by multiplying the scores for intensity and proportion, ranging from 0 to 12. For ICAM-1, at least 500 tumor cell for each xenograft sample (n = 5) were randomly selected and counted. The number of positive cell was counted and the positive index was calculated as follows: ICAM-1 index = (number of stained cells/total cell number) × 100%.
Statistical analysis
All experiments were performed at least in triplicate. All data are reported as the mean ± standard deviation. Using Excel 2007 (Microsoft Corporation, Redmond, WA, USA). Microsoft Office Excel 2007 (Microsoft Corporation, Redmond, WA, USA) and the statistical software SPSS13.0 (SPSS Inc., Chicago, IL, USA) were used in data processing and analyzing the significance with the one-way ANOVA,t-Test,or the log rank test (for Kaplan-Meier plots). Results with P < 0.05 were considered statistically significant.
Discussion
Various regulators are involved in the processes of malignancy and metastasis. Recent evidence suggests that proteins of the WAP family play an important role in tumor progression, malignancy, and metastasis [
9,
18].
WFCD2 is one of the members of the WAP family and previous research has shown that the blood concentration of
WFCD2 is higher in patients with ovarian cancer than in women with either healthy ovaries or benign ovarian tumors [
8,
19,
20]. Thus we hoped to identify the role of
WFCD2 in the malignancy and metastasis of ovarian cancer. In the present study, we analyzed the expression of
WFCD2 in ovarian cancer cell lines HO8910 and HO8910PM. The latter is derived from HO8910 and is considered to have more potency in invasion and metastasis than its parent HO8910 cell lines. We observed a higher expression of
WFCD2 in both the RNA and protein level in HO8910PM cells than that in HO8910 cells. These data indicate that
WFCD2 may be a tumor-specific gene involved in the malignancy and metastasis of ovarian cancer. A comparable correlation between
WFCD2 expression and the malignant behavior of ovarian carcinoma cells has also been established.
Our results shows that the high expression of WFCD2 is ovarian cancer tissues of all FIGO stages, and positively correlated to lymph node metastasis (p < 0.05) and implanted metastasis (p < 0.05), which indicates that high expression of WFCD2 may relate to the progression of ovarian cancer. Hence, WFCD2 may be a potential biomarker of clinical staging and may possibly be a biomarker for prognosis assessment. However, the application value of clinical examination still needs further evaluation.
After knockdown of WFCD2 expression, the invasion and migration rate was significantly lower in the WFCD2 cells compared to the blank control both in HO8910 and SKOV3 cells. The decreased malignancy of these ovarian cancer cells was confirmed to be associated with the lower levels of WFCD2. This suggests that WFCD2 accelerates the migration and invasion of ovarian cancer cells, as expected.
Unlike most solid tumors, ovarian cancer spreads mainly via implantation within the peritoneal cavity, and hematogenous metastasis is seldom observed [
18,
21‐
23]. The invasive and migratory capacity of ovarian cancer cells plays a key role in the metastasis process. To determine if the changes observed in vitro as a result of
WFCD2 knockdown are reproducible in vivo, we established an ovarian cancer xenograft model. This in vivo study using
WFCD2 gene-knockdown ovarian cancer cell HO8910–209 showed that
WFCD2 knockdown suppressed both ovarian tumor growth and peritoneal dissemination. The type of destination organs, the number of metastases, and the amount of nodules in HO8910–209 groups were significantly lower than in the control groups. These results are consistent with the results in vitro, and confirm that
WFCD2 knockdown inhibits cell migration and invasion, thereby inhibiting the malignancy and metastasis of ovarian cancer.
Many factors can affect the tumour metastasis. Our previous study indicated that knockdown of
WFCD2 induced cell apoptosis and depressed cell proliferation [
6]. In this study, several biochemical markers used to characterize metastases had been evaluated by immunohistochemical methods to further elucidate the role of
WFCD2 in tumorigenicity in vivo. In these biochemical markers, such as ICAM-1, VCAM-1, CD44, MMP2, MMP9, we observed that the expression of CD44, MMP2 and ICAM-1 was significantly reduced in
WFCD2 knockout tumor cells, which might explain WFCD2 knockdown reduced the mobility of tumor cells both in vitro and in vivo (Results are schematically summarized in Fig.
5f). ICAM-1 is an important cell-adhesion molecule directly linked to ovarian tumor growth, metastasis and chemo-resistance [
24]. CD44 is a receptor for
hyaluronic acid,up-regulation of CD44 represents a crucial event in the development of metastasis, recurrence, and drug resistance to current treatments in ovarian cancer. MMP-2 (along with
MMP-9) is capable of degrading
type IV collagen, the most abundant component of the
basement membrane. The interaction of MMP2 and CD44 is an important factor in selectively regulating the tumor microenvironment to promote tumor cell metastasis and is considered to be an inducer of EMT [
22,
25,
26]. To be interesting, Hokins etl had also reported that paracrine SLPI secretion upregulated MMP2 and MMP9 transcription and secretion in some cancer cells [
24,
25]. All this suggests a role for
WFCD2 in rebuilding the tumor microenvironment by regulating the expression of MMP2 and CD44. As both CD44 and MMP2 are inducer of epithelial-mesenchymal-transition (EMT),which strengthens our confidence that
WFCD2 might participate in tumor metastasis and disease processes by regulating the progression of EMT in ovarian cancer cells. However, the role of
WFCD2 as a regulator in EMT is still required further evaluation.
Acknowledgments
This work was supported by grants from the National High Technology Research and Development Program of China (863 Program) (No. 2012AA020205). This work was also supported by Guangzhou major collaborative innovation research projects (201508020052).