SerpinE2,a poor biomarker of endometrial cancer,promotes the proliferation and mobility of EC cells

Abstract

The SerpinE2 pathway is evolutionarily conserved and plays an important role in tumorigenesis. SerpinE2 (a small ubiquitin-related modifier), like ubiquitin, conjugates SerpinE2 proteins onto lysine residues of target proteins. SerpinE2 over-expression has been found in several tumors. Here, we detected the level of SerpinE2 in 72 samples of EC tissue using immunohistochemistry to assess the role of SerpinE2 in EC prognosis. Meanwhile, we knocked down SerpinE2 by siRNA in the HTB-111 and Ishikawa EC cell lines and analyzed the viability and mobility change using an MTT assay, an annexin V/PI apoptosis assay, a wound scratch test and a transwell assay. A Kaplan-Meier analysis indicated a negative correlation between the level of SerpinE2 and the EC prognosis. Silencing SerpinE2 induced cell apoptosis and reduced the migration ability. Our data suggest SerpinE2 works as an oncogene in EC.

1. Introduction

Endometrial cancer (EC) is one of the most common malignancies among females in both developed and developing countries. With the increase in obesity, improper application of hormone replacement therapy and aging populations, the incidence of EC has risen [1]. A noticeable symptom, vaginal bleeding, presents at an early stage in most EC patients, drawing their attention and generally resulting in a favorable prognosis. Although extremely rare, some patients present with primary bone pathology without vaginal bleeding [2]. In addition, up to 30% of EC patients were high-risk at the first visit, with the EC invading deep into the myometrium and showing local or extra pelvic metastasis [3]. For them, the cancer progresses more quickly and is resistant to contemporary therapy, and the prognosis is poor, with a five-year survival rate less than 50% [4]. Currently, selecting the optimized scheme for EC patients only depends on the surgical tumor stage (pTNM-system). For monitoring the response to therapy and detecting recurrent EC, CA125 and serum epididymis protein 4 (HE4) have been used as routine useful indices, and TP53, PTEN and PIK3CA might not be sufficient [5]. New predictive and prognostic biomarkers are urgently required.

Serine protease inhibitor E2 (SerpinE2), named glia-derived nexin or protease nexin-1, the phylogenetically closest relative of PAI-1, was first found to be secreted by fibroblasts and combined with proteases, such as thrombin, urokinase, and plasmin, to form complexes with certain serines [6]. Recently, SerpinE2 has attracted the attention of researchers because it targets the extracellular matrix and promotes the metastasis of malignant cells. SerpinE2 over-expression has been found to be a potential novel biomarker in many kinds of tumors [7]. For EC, the role of SerpinE2 has not been reported. Here, we measured the SerpinE2 level in 72 cases of EC using IH and analyzed its clinical significance. Moreover, we also knocked it down in an EC cell line to further verify its function.

2. Materials and methods

2.1 Patients and tissue samples

This study was approved by the Research Ethics Committee of the First Affiliated Hospital of Jinan University and the Nanfang Hospital. Written informed consent was obtained from all of the patients. Paraffin sections were obtained from 72 patients who were diagnosed with EC and enrolled at the department of Gynecology and Obstetrics Department of First Affiliated Hospital of Jinan and Nanfang Hospital the University between 2006 and 2010. The diagnosis of EC was confirmed based on FIGO guidelines (2009) and the 2008 WHO classification criteria. All of these patients underwent molecular and phenotypic classification and were distinguished as Types I (mainly endometrioid) and II (non-endometrioid). The follow-up was performed at 60 months by mail or phone.

2.2 Immunohistochemistry

IH was performed on EC paraffin sections using an Enhanced Polymer EliVision DAB Detection Kit (purchased from Maixin Biocompany, Fuzhou, China). Briefly, the paraffin-embedded tissue slides were heated, deparaffinized, and dehydrated, and then non-specific action was blocked by H ${}_2$ O ${}_2$ and a non-specific serum after a microwave antigen-retrieval procedure. The paraffin sections were incubated with a polyclonal primary rabbit antibody against SerpinE2 (1:50; Gene Tex, USA). An isotype-matched antibody was used as a negative control. Immunohistochemical scoring was performed without prior knowledge of the clinical response. Tissue sections were observed under an AX10-Imager A1 (ZEISS), and all pictures were captured using AxioVision 4.7 microscopy software. The sections were semi-quantitatively and manually scored for positive brown particles. The dominant staining intensity in tumor cells was scored as 0 $=$ negative; 1 $=$ weak (0 $\sim$ 25%); 2 $=$ intermediate (26 $\sim$ 50%); 3 $=$ strong (51 $\sim$ 75%); or 4 $=$ extra strong (76 $\sim$ 100%). We calculated the dominant staining intensity from five views and added the scores together.

2.3 Cell culture and siRNA synthesis

HTB-111 and Ishikawa cells were maintained in DMEM medium (Gibco, Carlsbad, CA, USA) containing 10% heat-inactivated fetal bovine serum albumin (Logan, USA) in a 95% humidified incubator with 5% CO ${}_2$ at 37 ${}^{\circ }$ C. siRNA of SerpinE2 was purchased from Jima Com, and the sequence was 5’-CUGGGAAUGGAGGAAGAAG-3’.

2.4 Cell viability assay

The cells were seeded into 96-well culture plates at a density of 1.5 $\times$ 10 ${}^4$ cells/well in 100 $\mu$ L completed medium for 12 h. Then, the medium was changed to FBS-free DMEM medium for 24 h. For the control group, FBS was added to a final concentration of 10%. For the experimental group, siRNA-SerpinE2 was transfected with Lipofectamine 3000 reagent according to the directions for 6 h, and the medium was changed to new DMEM medium with 10% FBS. The final volume of the two groups was 100 $\mu$ L. Viable cells were measured with methylthiazolyldiphenyl-tetrazolium bromide (Sigma Aldrich, St. Louis, MO) 24 h later according to the manufacturer’s instructions. The optical density of each well was read at 490 nm using a microplate reader (ELX800, Bio-Tek, USA). The inhibition ratio of proliferation was assessed with the following equation: inhibition ratio $=$ 1-(Experimental/Untreated Control).

2.5 Cell apoptosis assay

The cells were seeded into 6-well culture plates. The transfer process was almost performed as described above, except the amount of Lipofectamine 3000 reagent differed according to the protocol (different wells were given different amounts of DNA and transfer reagent). After transfection for 24 h, both suspended and adherent cells were collect and labeled with FITC-labelled annexin V and propidium iodide (PI) (BD Bioscience, USA). A FACScan instrument (Becton Dickinson, USA) was used to count the cells (1 $\times$ 10 ${}^3$ ) at an excitation wavelength of 490 nm. CellQuest software was used for data collection and processing.

2.6 Wound-healing assay

The scratch wound assay was performed to detect the mobility of EC cells. HTB-111 and Ishikawa cells were seeded in six-well plates overnight; then, the cells were transfected with the siRNA-SerpinE2 or the NC control for 6 h. The cells were incubated in fresh DMEM complete medium overnight. Then, the confluent cell monolayer was scraped using a 200 $\mu$ L sterile pipette tip. After cells were washed with PBS twice, they were cultured with new DMEM medium (including 10% FBS with or without IC50 Selumetinib). Photo images of the plates were captured 48 h later.

2.7 Migration assay

After cells were transfected with siRNA-SerpinE2 for 6 h, they were digested with 0.25% trypsin and then seeded into the upper layer of a Boyden chamber (Haimen, Jiangsu province, China). After incubating for 8 h, non-migratory cells were removed from the top well. The cells were fixed with 4% paraformaldehyde and stained with crystal violet. Photo images of the plates were photographed.

2.8 Matrix metalloprotein (MMP) activity ELISA assay

Using a total MMP-2 and MMP-9 Quantikine ELISA Kit assay (R&D Systems, USA), we detected the activity of MMP-2 and MMP-9 according to the manufacturer’s instructions. After transfection of siRNA for 24 h, the cells were washed with serum-free medium. The medium was collected 24 h later and centrifuged at 500 rpm for 10 min. The MMP-9 antibody or MMP-2 antibody was coated on the 96-well plate, and the supernatant was added to plate and incubated at 4 ${}^{\circ }$ C overnight. The plated was washed with buffer 3 times, and 50 $\mu$ l assay buffer and 50 $\mu$ l detection buffer were added to the wells in that order. After incubation at 37 ${}^{\circ }$ C for 1 h, the OD value at 405 nm was measured using a Microplate Reader (Bio-Tek).

2.9 Statistical analysis

One-way ANOVAs were used to identify the impact of clinicopathological factors and SerpinE2 expression in EC patients. Patient survival rates were calculated using the Kaplan-Meier method, and statistically significant differences in survival were identified using the log-rank test. All P values were two-tailed. Values of $P<$ 0.05 were considered significant. All analyses were performed using SPSS software version 19.0.

3. Results

3.1 Association of SerpinE2 expression with clinicopathological characteristics of EC patients

Table 1

Relationship between SerpinE2 expression and clinical features in endometrial cancer ( $n=$ 72)

Patient characteristics	Low SerpinE2	High SerpinE2	P value
	group	group
Age
$<$ 55	25	11	0.804
$⩾$ 55	24	12
Lymph node metastasis
Negative	34	10	0.036
Positive	16	13
Distant metastasis
Negative	41	13	0.013
Positive	8	10
Vessel invasion
Negative	33	9	0.023
Positive	16	14
ER
Negative	10	10	0.042
Positive	39	13
PR
Negative	28	10	0.285
Positive	21	13
Pathology
Adenocarcinoma	44	20
Non-adenocarcinoma	5	3
Stage
I	15	1	0.020
II	28	17
III/IV	6	5
Histological grade
G1	21	7	0.067
G2	27	12
G3	1	4

Figure 1.

Expression of SERPINE2 in human EC tissue and its clinical value. (A) Immunohistochemical staining of SerpinE2 was performed, and representative images of weak or moderate staining (a and c) and strong positive staining (b and d) are shown. a) and b) are at 200 $\times$ optical magnification; c) and d) are at w00 $\times$ optical magnification. (B) IHC analysis of SerpinE2 expression in the low and high expression group. (C) The Kaplan-Meier survival curve showed a correlation between the SerpinE2 level and overall survival.

A total of 72 patients were enrolled in this study. Among them, 36 (36.11%) were $<$ 55 years old, and 61 (84.72%) were stage I–II. The detailed clinical features of the enrolled patients are summarized in Table 1. We identified the SerpinE2 level by IH. Figure 1A presents representative images of the immunostaining showing the positive brown particles in the plasma. Panels a and c show less intense, mostly weak to moderate staining compared to the representative images showing strong positive staining of cancer cells in panels b and d. Accordingly, the median fold change of SerpinE2 was used as a cutoff value to divide all 72 patients into two groups (Fig. 1B): the low expression group ( $n=$ 49) and the high expression group ( $n=$ 23). As shown in Table 1, high SerpinE2 expression was closely correlated with lymph node metastasis ( $P=$ 0.036), distant metastasis ( $P=$ 0.013), vessel invasion ( $P=$ 0.023), ER ( $P=$ 0.042), and advanced TNM stage ( $P=$ 0.020). By contrast, there was no association between SerpinE2 expression and other clinical factors, including age, PR, grade and pathology (all $P>$ 0.05).

3.2 High levels of SerpinE2 are associated with poor prognosis of EC patients

Using the Kaplan-Meier analysis, we compared the difference in the survival ratio between low and high SerpinE2 expression in EC (Fig. 1C). Among the 23 patients with high SerpinE2 expression, 9 patients died (60.9%). Among those with low SerpinE2 expression, 9 patients also died (81.6%, 9/49). When comparing the 5 year overall survival rate of the two groups, there was a significant difference between the high and the low SerpinE2 expression group ( $P=$ 0.045).

3.3 The effect of SerpinE2 on the viability of EC cells

After a 24 h transfection of siRNA-SerpinE2 into EC cells, we measured the mRNA and protein levels of SerpinE2. The QRT-PCR results showed that siRNA reduced the SerpinE2 level to one third of the control levels (Fig. 2A). The gray value of the WB analysis showed that the protein level in the siRNA group was half of that in the NC group (Fig. 2B). To determine the effect of siRNA-SerpinE2 on the proliferation of HTB-111 and Ishikawa cells, an MTT test was performed. The results showed that the proliferation ratio of the siRNA group was 70–75% of that of the NC group in both the HTB-111 and Ishikawa cells (Fig. 2C). The apoptosis assay indicated that siRNA-SerpinE2 significantly induced apoptosis (Fig. 2D). Bajou reported that the anti-apoptosis factor, Bcl-2, is negatively regulated by SerpinE2 [8]. We clarified this by QRT-PCR and WB. siRNA-SerpinE2 up-regulated expression of Bcl-2 at both the mRNA and protein level (Figs 2E and F).

Figure 2.

SerpinE2 took part in cell proliferation. (A) After transfection of siRNA-SerpinE2 into HTB-111 and Ishikawa cells for 24 h, we detected the SerpinE2 mRNA levels by QRT-PCR. Compared with the SerpinE2 levels in the untreated control and scrambled siRNA (NC) control groups, siRNA-SerpinE2 sharply decreased SerpinE2 expression ( ${}^{*}$ indicates $P<$ 0.05). (B) Meanwhile, WB analysis was performed; in both HTB-111 (left panel) and Ishikawa (right panel) cells, the protein level of SerpinE2 in the target siRNA group was only half of that of the other two controls. (C) The inhibitory effect of siRNA-SerpinE2 on the proliferation of the EC cell lines was examined by MTT assay. The bar graph demonstrates that the viability of HTB-111 and Ishikawa cells in the siRNA-SerpinE2 group was nearly three quarters of control. (D) The quantitative apoptosis data also supported the above results; in the siRNA-SerpinE2 group, the apoptosis ratio was 20–25%. For the control groups, it was less than 10%. (E) After HTB-111 and Ishikawa cells were transfected with siRNA-SerpinE2 for 24 h, we detected the Bcl-2 mRNA level by QRT-PCR. Compared with the observed levels in the untreated control and scrambled siRNA (NC) control groups, siRNA-SerpinE2 sharply increased Bcl-2 expression ( ${}^{*}$ indicates $P<$ 0.05). (B) The WB results showed the same tendency as the mRNA results; the protein level of Bcl-2 was significantly higher in the siRNA-SerpinE2 group than the levels in the two control groups.

3.4 The effect of SerpinE2 on the mobility of EC cells

Furthermore, we also measured the mobility change in EC cells after knock-down of SerpinE2 expression using the wound-healing assay. After cells were transfected for 48 h, the percentage of the wound healing area in the siRNA group was significantly less than that of the control group (Fig. 3A). The transwell assay revealed that there were fewer EC cells in the siRNA group than in the NC and untreated control group (Fig. 3B). The activity of MMP-2 and MMP-9 were both decreased in the siRNA-SerpinE2 group by approximately 65–70% of control ( ${}^{*}$ indicates $P<$ 0.001, Fig. 3C).

Figure 3.

The mobility of EC cells was reduced with suppression of SerpinE2. (A) After HTB-111 and Ishikawa cells were transfected with siRNA-SerpinE2 for 48 h, the wound-scratch assay demonstrated that siRNA-SerpinE2 suppressed the healing of the EC cells; the wounded area was twice the size of that in the control groups. (B) The transwell invasion assay demonstrated that the invasive capacity of EC cells was reduced with siRNA-SerpinE2 treatment. (C) After HTB-111 and Ishikawa cells were transfected with siRNA-SerpinE2 for 48 h, the supernatant was collected to detect the MMP2 and MMP9 activity by ELISA. The mean absorbance value of MMP-2 and MMP-9 in the siRNA-SerpinE2 group was 35–40% of that of control ( ${}^{*}$ indicates $P<$ 0.05).

4. Discussion

Protein inhibitors are the key factors in the maintenance of protease activity, which is the last step in protease activity regulation. SERPINs are a superfamily of widely distributed and structurally related serine and cysteine protease inhibitors that regulate fibrinolysis, coagulation, complement activation, inflammation, cell mobility, cellular differentiation and apoptosis processes. SERPINs always form tight but reversible binary complexes that interact with proteases as inhibitors, substrates, or both and trigger conformational changes of both SERPINs and proteases [9]. SerpinE2/PN-1, unlike any other circulating SERPIN, is barely detectable in plasma but is expressed in tissues and mainly takes part in the regulation of extracellular matrix destruction [10]. Because of this feature, SerpinE2/PN-1 has been studied in the vasculature, nervous system and the behavior of cancer cells. Anoikis is described as apoptosis of adherent cells induced by the disruption of integrin-mediated cell-matrix interactions. Anoikis resistance is the key process of metastasis, and anti-apoptosis is its first characteristic. Numerous studies have suggested that SerpinE2 is required for over proliferation, anti-apoptosis and malignant transformation of tumor cells [11, 12]. Rossignol reported that over-expressed SerpinE2/PN-1/PAI-1 in Chinese hamster ovary fibroblast cells significantly inhibited the activity of plasmin and tissue-type plasminogen activator via the formation of inhibitory complexes and prevented cell detachment and apoptosis [13]. Rømer found that SerpinE2/PN-1/PAI-1 influences sensitivity to etoposide-induced apoptosis through the PI3K/Akt cell survival pathway by acting upstream of PI3K and Akt [14]. Bajou reported that SerpinE2/PN-1/PAI-1 protects EC cells from Fas/FasL-mediated apoptosis through Bcl-2 [8]. In our paper, our results also supported this conclusion. Silencing of SerpinE2 induced inhibition of viability and triggered apoptosis in EC cell lines. In addition, a large body of work has revealed that SerpinE2 is deeply involved in tumor metastasis. Smirnova found that blocking SerpinE2 caused a reduction in M2-like TAMs, diminished CCL2 and changed the collagen deposition, fiber structure and organization. SerpinE2 is one of the most important factors in tumor cell dissemination. Wu described that SerpinE2 promotes melanoma metastasis through the glycogen synthesis kinase 3 $\beta$ , GSK-3 $\beta$ , signaling pathway [15]. SerpinE2 has been shown to be highly expressed in osteosarcoma tissue, stimulates osteosarcoma cell proliferation, and promotes drug resistance [16]. SerpinE2 is also a poor biomarker for prostate cancer [17], gastric cancer [18], colorectal cancer [19], etc. Here, we first evaluated the impact of SerpinE2 protein expression on the survival of EC patients. Using IH analysis, the expression of SerpinE2 in EC tissue was calculated by both staining intensity and the percentage of positive cells. The SerpinE2 level was found to be correlated with a poor prognosis. There was a significant difference in the 5 year overall survival rate between the low and high SerpinE2 groups: 81.6% vs 60.9%, respectively ( $P=$ 0.045). Our results also indicated that high expression of SerpinE2 was positively associated with metastasis of EC including lymph node metastasis ( $P=$ 0.036), distant metastasis ( $P=$ 0.013), and vessel invasion ( $P=$ 0.023). Metastasis is the most common cause leading to death in EC. The initial stage of metastasis is extracellular matrix remodeling to overcome tissue barriers. The proteolytic enzymes, matrix metalloproteinases (MMPs), play the pivotal role in pericellular matrix degradation during basement membrane and interstitial tissue transmigration programs. Recent data have indicated that SerpinE2 affects malignant cell migration and invasiveness through the regulation of MMP expression [20]. In this paper, we also examined the effects of SerpinE2 on the mobility and activity of MMP-2 and MMP-9 of EC cells. The wound-healing and transwell assays showed that after knocking down SerpinE2, the mobility of EC cells was significantly reduced along with the inactivation of MMP-2 and MMP-9. The activity of MMP-2 and MMP-9 in the siRNA-SerpinE2 group was almost one third of that of the control group.

Our findings suggest that aberrant expression of SerpinE2 contributes to tumorigenesis in EC through the promotion of cancer cell proliferation and invasion. SerpinE2 may play important roles in the development of EC and work as a valuable marker for metastasis and, thereby, prognosis for EC patients.