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Keywords:

  • endometrial cancer;
  • HAI-1;
  • HAI-2;
  • tumor suppressor gene;
  • favorable prognosis marker

Abstract

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Hepatocyte growth factor activator inhibitors (HAI-1 and HAI-2) are Kunitz-type serine protease inhibitors that have a broad inhibitory spectrum against serine proteases. This is the first study to investigate the role of HAI-1 and HAI-2 in endometrial cancer. We investigated the biological functions of HAI-1 and HAI-2 using KLE and HEC-251 endometrial cancer cell lines, thus HAI-1 and HAI-2 were examined in uterine normal endometrium, endometrial hyperplasia and cancer specimens by immunohistochemistry. HAI-1 and HAI-2 showed potential inhibitory effects on cell proliferation, migration and cellular invasion by reduction of matriptase and hepsin expression. This in turn led to an increase in the levels of E-cadherin and Slug, and a reduction in the levels of Vimentin, SIP1, Snail and Twist, and hence ER and PR signal transduction in endometrial cancer cells. The levels of HAI-1 and HAI-2 expression were significantly decreased in endometrial cancer specimens relative to the corresponding normal endometrium specimens. Low HAI-1 and HAI-2 expression was a significant predictor for a poor prognosis compared with high HAI-1 and HAI-2 expression. These findings indicate that HAI-1 and HAI-2 could be considered as therapeutic targets and used as favorable prognosis markers for endometrial cancer.

Endometrial cancer is the most common gynecologic malignancy in the world.1 In Japan, endometrial cancer is currently the fourth most common gynecologic malignancy in women, with an estimated incidence of 5,005 new cases in 2007.2 The incidence of endometrial cancer has been increasing markedly in recent years. Because of the poor prognosis of endometrial cancer, several studies have been carried out to develop more effective treatments for this disease. Along with surgery, chemotherapy has emerged as one of the leading contenders in the treatment armamentarium. It is now widely accepted that new approaches for the treatment of endometrial cancer are pivotal to further improve the prognosis of this disease.

The metastatic process involves degradation of the extracellular matrix (ECM) including interstitial basement membrane by proteinases that facilitate cell detachment, local and systemic spreading. Hepatocyte growth factor (HGF) is secreted from mesenchymal cells of the liver as an inactive single-chain form and normally remains in this form when associated with the ECM.3 HGF induces proliferation, motility and morphogenesis of epithelial and endothelial cell types via a high affinity receptor tyrosine kinase, c-Met.4, 5 High c-Met overexpression may be associated with poor prognosis of endometrial cancer.6 HGF is secreted as an inactive pro-peptide, which is cleaved by HGF activator (HGFA) into its active form. HGFA is regulated by two inhibitors, HGFA inhibitor type I (HAI-1) and type II (HAI-2), both type I transmembrane glycoproteins with two Kunitz-type serine protease domains in their extracellular portion and are expressed on the surface of epithelial cells.7–10 To date, several studies on HAI-1 and HAI-2 expression in tumor tissues have been published. Previously, we reported that HAI-1 and HAI-2 levels are significantly decreased during the progression of cervical and ovarian cancer.11–13 Recent studies reveal that the reduced expression of HAI-1 is possibly involved in the progression of prostate, breast and gastric cancer.14–16 In addition, the downregulation of HAI-2 in glioblastomas, hepatocellular and renal carcinomas was partly due to the hypermethylation of the HAI-2 promoter region.17–19 HAI-1 and HAI-2 potently inhibit a variety of serine proteases that may be involved in carcinogenesis, invasion and metastasis. HAI-1 and HAI-2 appear to be the cognate inhibitors of matriptase and hepsin and belong to the type II transmembrane serine protease superfamily, and prostasin is a glycosylphosphatidylinositol-anchored membrane serine protease protein.7–11 Matriptase and hepsin could be important tools for identifying patients with poor prognosis of endometrial cancer.20, 21

Epithelial-mesenchymal transition (EMT) is an essential morphologic conversion occurring during embryonic development. Increasing evidence suggests that a similar process occurs during cancer progression when tumor cells acquire the capacity to migrate, invade and metastasize. A key feature in the initiation and execution of EMT is the downregulation of E-cadherin expression.22–24 The activation of these E-cadherin repressors may be induced by signaling mediated by growth factors, such as HGF, insulin-like growth factor (IGF), epidermal growth factor (EGF), fibroblast growth factor (FGF) and transforming growth factor β (TGF-β) via their specific receptors.25–27 The transcriptional repressors of E-cadherin are zinc finger transcription factors, including Snail, Slug, Smad-interacting protein 1 (SIP1) and Twist, which is also implicated in this process.28–31 Cheng et al. have reported that HAI-1 acts as a novel regulator of E-cadherin and that its inactivation plays a role in inducing EMT of carcinoma cells.32 However, it is not clear how the mechanisms of action of HAI-1 and HAI-2 are involved in endometrial cancer, or how their roles are intertwined. The aim of this study was to determine whether HAI-1 and HAI-2 could be considered as therapeutic targets for treatment and used as favorable prognosis markers of endometrial cancer.

Material and Methods

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Cell culture, media and generation of transfectants

KLE (ATCC number: CRL-1622) and HEC-251 (Japanese Collection of Research Bioresources (JCRB) number: JCRB1141) cell lines were derived from human endometrial cancers. The KLE cell line was maintained in Dulbecco's modified Eagle's medium (DMEM)/F12 medium (Life Technologies, Grand Island, NY), supplemented with 10% fetal bovine serum (FBS). The HEC-251 cell line was maintained in DMEM (Life Technologies, Grand Island, NY) with 10% FBS. A cDNA encompassing the whole coding region of HAI-1 or HAI-2 was constructed by polymerase chain reaction using full-length HAI-1 cDNA or HAI-2 cDNA as a template. Primers used were: 5′-TT-GGAATTCGCGATGGC CCCTGCGAGGAC-3′ and 5′-TTAGACTCAGAGGGGCCG GGTGGTGT-3′ for HAI-1, and 5′-AGCTCTAGAGCCATG GCGCAGTGCGG-3′ and 5′-TTAGTCGACTCACAGGACAT ATGTGTTCTTC-3′ for HAI-2. The polymerase chain reaction products were subcloned into the EcoRI/SalI site (HAI-1) or the XbaI/SalI site (HAI-2) of the expression plasmid pCIneo (Promega, Madison, WI), as described previously.33 The HAI-1 and HAI-2 cDNA expression vectors were transfected into each cell line using the TransFast transfection reagent (Promega, Madison, WI). Mock transfected cells served as a control.

Western blotting analysis

Cell lysates were collected and estimated using a Protein Assay system (Bio-Rad, Hercules, CA) according to the manufacturer's protocols. Proteins from each cell line were subjected to SDS-PAGE, and then were transferred onto a nitrocellulose membrane. Polyclonal and monoclonal antibodies used for immunoblotting were as follows: HAI-1 and HAI-2 (Santa Cruz Biotechnology, Santa Cruz, CA), and β-actin (Sigma Chemical, St. Louis, MO). Working dilution of all primary antibodies was 1:1,000. Membranes were then incubated with the appropriate secondary antibodies. Expression levels of antigen–antibody complexes were detected with an enhanced chemiluminescence kit (Amershan Biosciences, Piscataway, NJ).

MTS and cell viability assays

To evaluate the effects of HAI-1 and/or HAI-2 on cell proliferation, MTS (Promega, Madison, WI) and cell viability assays (Invitrogen, Eugene, OR) were performed. For the MTS assay, the cells were seeded into 96-well plates and transfected when the cell density reached 5 × 104 cells per well. Cells were transiently transfected with either HAI-1 vector or HAI-2 vector, or both HAI-1 and HAI-2 vectors for 12, 24, 36 and 48 hr and then MTS was added for 1 hr. The absorbance was read at 490 nm using an ELISA plate-reader (Bio-Rad Systems, Hercules, CA). The cell viability was analyzed using SYTO 10 green fluorescent nucleic acid stain and dead red (ethidium homodimer-2) nucleic acid stain (Live/Dead® reduced biohazard viability/cytotoxicity kit). Cells were transiently transfected with either HAI-1 vector or HAI-2 vector, or both HAI-1 and HAI-2 vectors for 48 hr and then SYTO 10 green fluorescent nucleic acid stain and dead red nucleic acid stain were added to each well and incubated for 15 min. Cell fluorescence was observed using a fluorescent microscope (Olympus, Tokyo, Japan).

Motility invasion assay

To investigate the effects of HAI-1 and/or HAI-2 on cell motility invasion, we used monolayer wounding (scratch) and chemotaxicells (polycarbonate filter, pore size 8 μm; Kurabo, Osaka, Japan) coated with type IV collagen. For the monolayer wounding assay, cells were allowed to form a monolayer on a culture dish, and a wound was made by scratching the monolayer with a pipette tip. After the scratched cells were removed and transiently transfected with either HAI-1 or HAI-2 vector, or both HAI-1 and HAI-2 vectors, the cells were cultivated for 24 hr. Using chemotaxicells coated with type IV collagen, cells (1 × 105) in 100 μL of DMEM/F12 or DMEM medium and 0.1% bovine serum albumin were placed in the upper compartment and transiently transfected with either HAI-1 or HAI-2 vector, or both HAI-1 and HAI-2 vectors, and then the cells were incubated for 48 hr. After incubation, the cells on the upper surface of the filter were wiped off with a cotton swab. The cells on the lower surface were stained with hematoxylin and counted in 10 randomly selected fields.

Matrigel invasion assay

To investigate differences in the matrigel invasive ability between cells expressing HAI-1 and HAI-2, we used the BD BioCoat Matrigel Invasion Chamber (BD Bioscience, Bedford, MA). The KLE and HEC-251 cells transiently transfected with either HAI-1 or HAI-2 vector, or both HAI-1 and HAI-2 vectors were added in situ with 10 μg/mL of DiI (Invitrogen, Carlsbad, CA) in DMEM/F12 or DMEM with 10% FBS for 1 hr. Cells (5 × 104) of each genotype were added to inserts, and 0.75 mL of medium was added to the bottom of each well. After 48 hr of incubation, membranes were removed from the insert and mounted on slides, and then invading cells were counted under the microscope. Matrigel assays were performed in triplicate.

Reverse transcription-polymerase chain reaction

Total RNA was extracted from cell lines using the acid guanidium-phenol-chloroform method (ISOGEN, Nippon Gene, Tokyo, Japan) according to the manufacturer's instructions. The PCR products were analyzed using 1.5% agarose gel electrophoresis. As the internal control, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA was also measured using quantitative reverse transcription-polymerase chain reaction (RT-PCR). The primers for HAI-1, HAI-2, matriptase, hepsin, prostasin, E-cadherin, Vimentine, SIP1, Snail, Slug, Twist and GAPDH, were as described previously.15, 32, 34

Transient transfection assay of hormone responsive elements

Estrogen responsive elements (ERE) and progesterone responsive elements (PRE) were used for the estrogen and progesterone signaling reporter assays. The ptk-ERE-Luc and ptk-PRE-Luc were kindly provided by Dr. Hayashi S.35 The transient transfections were performed using lipofectamine™ 2000 (Life Technologies, Grand Island, NY). For the luciferase reporter assay, the cells were transfected with 0.5 μg of estrogen responsive plasmid or 0.5 μg of progesterone responsive plasmid in combination with 0.05 μg of pTK-RLUC (Promega, Madison, WI) as the internal control. After 48 hr of incubation, KLE and HEC-251 cells transiently transfected with either HAI-1 or HAI-2 vector, or both HAI-1 and HAI-2 vectors were harvested. Their proteins were then extracted using the Dual-Luciferase reporter assay system (Promega, Madison, WI). Firefly and renilla luciferase activities were both measured concurrently for 12 sec using a luminometer (LUMAT LB9507, Berthold, Germany). The assays were carried out using quadruplicate transfection experiments, and at least three independent values were analyzed to confirm reproducibility.

Cell growth in monolayer

For evaluation of cell growth in a monolayer, cells were plated at a density of 3 × 104 cells per well in 6-well plates containing DMEM/F12 or DMEM with 10% FBS. The cell number was counted in triplicate after 2, 4, 6, 8 and 10 days using a hemocytometer to assess cell proliferation.

Patients and tissues

Patients with normal endometrium (n = 20), endometrial hyperplasia (n = 10) (endometrial hyperplasia, simple (n = 1), endometrial hyperplasia, complex (n = 2), atypical endometrial hyperplasia, complex (n = 7)) and adenocarcinoma (n = 56) were treated at Okayama University Hospital between January 1996 and November 2004. Patients with distant metastasis were excluded from this study. Tumor specimens were obtained at the time of surgery and immediately fixed in 10% neutral-buffered formalin and embedded in paraffin. Histological cell types were diagnosed according to the WHO classification: 56 were classified as endometrioid adenocarcinomas. Histological grades were set according to the International Federation of Gynecology and Obstetrics (FIGO) staging classification as follows: 18 were grade 1, 23 were grade 2 and 15 were grade 3. Surgical staging was reviewed based on the FIGO staging system: 22 were allocated to stage I, 2 to stage II, 25 to stage III and 7 to stage IV. The median age at the time of surgery was 56 years (range, 34–81). Disease-free and overall survival rates were defined as the interval between the initial operation and either clinically or radiologically proven recurrence or death, respectively.

Immunohistochemistry and staining evaluation

Formalin-fixed, paraffin-embedded sections, 4-μm thick were deparaffinized with xylene and rehydrated in ethanol. Endogenous peroxidase activity was quenched by methanol containing 0.3% hydrogen peroxidase for 15 min. Then, sections were incubated at room temperature with anti-HAI-1 and anti-HAI-2 (Santa Cruz Biotechnology, Santa Cruz, CA) followed by staining using a streptavidin-biotin-peroxidase kit (Nichirei, Tokyo, Japan). The sections were counterstained with hematoxylin. The level of HAI-1 and HAI-2 staining in epithelial cells was classified into three groups by scoring the percentage of positive cells: strong (2); >50% of cells stained, moderate (1); 10–50% of cells stained, and weak (0); <10% of cells stained. Microscopic analyses were independently conducted by two independent examiners with no prior knowledge of the clinical data. Final decisions in controversial cases were made using a conference microscope.

Statistical analysis

Statistical analyses were performed using the Mann–Whitney U-test for comparison with the control and one-factor ANOVA followed by Fisher's protected least significance difference test for all pairwise comparisons. The analyses were performed with the software package StatView version 5.0 (Abacus Concepts, Berkeley, CA). Differences were considered significant at p < 0.05.

Results

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Protein expression and cell proliferation

We first examined the protein levels of HAI-1 and HAI-2 in human endometrial cancer cell lines. All of the six endometrial cancer cell lines (KLE, HEC-251, Ishikawa, HEC-1A, HEC-50B and RL95-2) showed remarkably low and undetectable levels of HAI-1 and HAI-2, as demonstrated by RT-PCR and Western blot analysis (data not shown). Among the endometrial cancer cell lines, we chose KLE and HEC-251 endometrial cancer cells for further study.

The expression of HAI-1 and HAI-2 protein levels were analyzed using transient transfection with either HAI-1 or HAI-2 vector, or both HAI-1 and HAI-2 vectors into KLE and HEC-251 endometrial cancer cell lines. As shown in Figure 1a, HAI-1 and HAI-2 protein levels were highly expressed in the cells transiently transfected with either HAI-1 or HAI-2 vector, or both HAI-1 and HAI-2 vectors into KLE and HEC-251 endometrial cancer cell lines.

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Figure 1. Evaluation of cells transiently transfected with either HAI-1 or HAI-2 vector, or both HAI-1 and HAI-2 vectors. (a) Western blot analysis of HAI-1 and HAI-2 expression after transient transfection with either HAI-1 or HAI-2 vector, or both HAI-1 and HAI-2 vectors in KLE and HEC-251 cells for 48 hr. β-actin antibody was used as the loading control in the same blot. (b) MTS assay of KLE and HEC-251 cells after transient transfection with either HAI-1 or HAI-2 vector, or both HAI-1 and HAI-2 vectors for 12, 24, 36 and 48 hr. The assays were carried out in quadruplicate. (c) The cell viability of KLE and HEC-251 after transient transfection with either HAI-1 or HAI-2 vector, or both HAI-1 and HAI-2 vectors for 48 hr. Cell fluorescence was evaluated using a fluorescence microscope. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

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Previous reports have shown that HAI-1 and HAI-2 inhibited cell proliferation in cervical and ovarian cancer.11–13 Hence, the cell proliferation after transient transfection with either HAI-1 or HAI-2 vector, or both HAI-1 and HAI-2 vectors into KLE and HEC-251 endometrial cancer cell lines examined by MTS and cell viability assays. The cells transiently transfected with either HAI-1 or HAI-2 vector, or both HAI-1 and HAI-2 vectors into KLE and HEC-251 cells for 48 hr were significantly inhibited as evaluated by the MTS assay (Fig. 1b). The percentages of viable cells were decreased to 64.8%, 55.9% and 50.1% (KLE), and 76.1%, 71.4% and 68.1% (HEC-251) of the control cell viabilities at 48 hr after transient transfection with either HAI-1 or HAI-2 vector, or both HAI-1 and HAI-2 vectors, respectively (Fig. 1c).

Motility invasiveness and matrigel invasion assessment

The purpose of these experiments was to study the role of endometrial cancers in the motility invasiveness and matrigel invasion of KLE and HEC-251 cells transiently transfected with either HAI-1 or HAI-2 vector, or both HAI-1 and HAI-2 vectors. Compared to the control, the motility invasiveness of the KLE and HEC-251 cells transiently transfected with either HAI-1 or HAI-2 vector, or both HAI-1 and HAI-2 vectors were much slower. Moreover, the percentages cells capable of motility invasiveness were decreased to 50.9% and 74.8%, 48.8% and 69.4%, 47.1% and 66.1% in a migration assay on type IV collagen at 48 hr after transient transfection with either HAI-1 or HAI-2 vector, or both HAI-1 and HAI-2 vectors into KLE and HEC-251 cells, respectively (Figs. 2a and 2b). These results suggest that HAI-1 and HAI-2 overexpression decreases cell adhesion and spreading. In addition, the percentages of cells capable of matrigel invasion were decreased to 50.5% and 67.7%, 45.3% and 63.8%, 41.4% and 57.2% in at 48 hr after transient transfection with either HAI-1 or HAI-2 vector, or both HAI-1 and HAI-2 vectors into KLE and HEC-251 cells, respectively (Fig. 2c). Both HAI-1 and HAI-2 have potential inhibitory effects on motility invasiveness and matrigel invasion in KLE and HEC-251 endometrial cancer cells.

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Figure 2. Effects on the motility and matrigel invasions of KLE and HEC-251 cells after transient transfection with either HAI-1 or HAI-2 vector, or both HAI-1 and HAI-2 vectors. (a) Cell scratch assay of KLE and HEC-251 cells after transient transfection with either HAI-1 or HAI-2 vector, or both HAI-1 and HAI-2 vectors for 24 hr. (b) Migration assay on type IV collagen of KLE and HEC-251 cells after transient transfection with either HAI-1 or HAI-2 vector, or both HAI-1 and HAI-2 vectors for 48 hr. (c) Matrigel invasion assay of KLE and HEC-251 cells after transient transfection with either HAI-1 or HAI-2 vector, or both HAI-1 and HAI-2 vectors for 48 hr. Following incubation, membranes were removed from the insert and mounted on slides. The numbers of invading cells were counted under the microscope. The motility and matrigel assays were performed in triplicate.

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RT-PCR assay

The proteinase activities have been implicated in EMT by activating growth factors and their receptors and cleaving cell–cell and cell–ECM adhesion molecules. Hence, we examined the serine protease and transcriptional repressors of EMT families after transient transfection with either HAI-1 or HAI-2 vector, or both HAI-1 and HAI-2 vectors into KLE and HEC-251 endometrial cancer cell lines by RT-PCR assay. The cells transiently transfected with either HAI-1 or HAI-2 vector, or both HAI-1 and HAI-2 vectors resulted in a significant increase in expression of HAI-1 and HAI-2 mRNA. The expression of matriptase mRNA was significantly decreased by transient transfection with either HAI-1 or HAI-2 vector, or both HAI-1 and HAI-2 vectors into KLE and HEC-251 cells. The expression of hepsin mRNA was significantly decreased by transient transfection with either HAI-1 or HAI-2 vector, or both HAI-1 and HAI-2 vectors into HEC-251 cells. The expression of hepsin mRNA was not detected in KLE cells with or without transient transfection of the HAI-1 and/or HAI-2 vector. However, the expression of prostasin mRNA was not related to the transient transfection with either HAI-1 or HAI-2 vector, or both the HAI-1 and HAI-2 vectors into KLE and HEC-251 cells. The transient transfection with either HAI-1 or HAI-2 vector, or both HAI-1 and HAI-2 vectors into KLE and HEC-251 cells caused a significant increase in the expression of E-cadherin mRNA. Moreover, the expression of Vimentin mRNA was also decreased accompanied by enhanced expression of E-cadherin mRNA by transient transfection with either HAI-1 or HAI-2 vector, or both HAI-1 and HAI-2 vectors into KLE and HEC-251 cells. The expression of SIP1 mRNA and Snail mRNA were significantly decreased by transient transfection with either HAI-1 or HAI-2 vector, or both HAI-1 and HAI-2 vectors into KLE and HEC-251 cells. In addition, the expression of Twist mRNA was significantly decreased by transient transfection with either HAI-1 or HAI-2 vector, or both HAI-1 and HAI-2 vectors into KLE cells. On the other hand, the expression of Slug mRNA was significantly increased by transient transfection with both HAI-1 and HAI-2 vectors into KLE cells (Fig. 3a).

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Figure 3. (a) PCR analysis of HAI-1, HAI-2, matriptase, hepsin, prostasin, E-cadherin, Vimentin, SIP1, Snail, Slug and Twist expression levels after transient transfection with either HAI-1 or HAI-2 vector, or both HAI-1 and HAI-2 vectors for 48 hr into KLE and HEC-251 cells. GAPDH was used as the loading control. (b) Transient transfection assays of estrogen responsive elements (ERE) and progesterone responsive elements (PRE) of KLE and HEC-251 cells after transient transfection with either HAI-1 or HAI-2 vector, or both HAI-1 and HAI-2 vectors for 48 hr. The assays were carried out in quadruplicate. (c) Monolayer growth of KLE and HEC-251 cells transiently transfected with either HAI-1 or HAI-2 vector, or both HAI-1 and HAI-2 vectors incubated for 2, 4, 6, 8 and 10 days in DMEM/F12 or DMEM medium supplemented with 10% FBS. The assays were carried out in triplicate.

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ER and PR signal transduction

Estrogen regulates the growth and development of the endometrium gland and is necessary for the maintenance of its functions.36 Estrogen receptor (ER) is activated not only by estrogen but also by growth factors.37, 38 Activated ER contributes to the proliferation, antiapoptosis and metastasis of tumor cells. This effect is the result of induction of its downstream genes whose promoter regions contain ERE.39, 40 We examined ERE and PRE signal transduction after transient transfection with either HAI-1 or HAI-2 vector, or both HAI-1 and HAI-2 vectors into KLE and HEC-251 cells using the luciferase reporter assay. ERE and PRE promoters were significantly decreased by transient transfection with both HAI-1 and HAI-2 vectors into KLE cells. In HEC-251 cells, ERE of transcriptional activities were significantly decreased by transient transfection with either HAI-1 or HAI-2 vector, or both HAI-1 and HAI-2 vectors. PRE of transcriptional activities were significantly decreased by transient transfection with either HAI-2 vector, or both HAI-1 and HAI-2 vectors into HEC-251 cells (Fig. 3b).

Cell growth in a monolayer

Effects of HAI-1 and HAI-2 expression on cell proliferation were analyzed using KLE and HEC-251 endometrial cancer cell lines transiently transfected with either HAI-1 or HAI-2 vector, or both HAI-1 and HAI-2 vectors. We found a significant inhibitory effect in the KLE cells transiently transfected with either the HAI-1 or HAI-2 vector, or both the HAI-1 and HAI-2 vectors compared to the control (Fig. 3c).

Human endometrium tissues by immunostaining

HGF-dependent activation may have a role in conventional endometrial tumorigenesis, thus the pro-HGF-activation machinery might be important, whereas the role of HAI-1 and HAI-2 in endometrial cancer tissues is not known. Expression levels of HAI-1 and HAI-2 were examined in human endometrial tissues by immunostaining; Figures 4a4f illustrate representative immunostaining patterns of HAI-1 and HAI-2. Weak epithelial staining was observed in 20 (23.3%, HAI-1) and 21 cases (24.4%; HAI-2), moderate staining in 18 (20.9%; HAI-1) and 25 cases (29.0%; HAI-2) and strong staining in 48 (55.8%; HAI-1) and 40 cases (46.6%; HAI-2). The mean scores of epithelial staining in HAI-1 or HAI-2 were 1.60 and 1.55 for normal human endometrium, 1.40 and 1.40 for hyperplasia and 1.21 and 1.07 for cancer samples. Interestingly, normal endometrium had the strongest expression of HAI-1 and HAI-2 compared with endometrial cancer samples as shown in Figures 4g and 4h (Mann–Whitney U-test, p < 0.05).

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Figure 4. Representative immunostaining patterns of hepatocyte growth factor activator inhibitors (HAI-1 and HAI-2). (a) Weak HAI-1 staining in endometrial cancer (endometrioid adenocarcinoma Grade 2). (b) Moderate HAI-1 staining in atypical hyperplasia. (c) Strong HAI-1 staining in endometrial cancer (endometrioid adenocarcinoma Grade 1). (d) Moderate HAI-2 staining in endometrial cancer (endometrioid adenocarcinoma Grade 3). (e) Moderate HAI-2 staining in endometrial cancer (endometrioid adenocarcinoma Grade 2). (f) Strong HAI-2 staining in endometrial cancer (endometrioid adenocarcinoma Grade 1) (original magnification, ×200). Histogram of (g) HAI-1 and (h) HAI-2 expression by specimens (uterine normal endometrium, endometrial hyperplasia and endometrial cancer). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

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Table 1 shows the distribution of cases scored as positive for each of the biological parameters examined, according to clinicopathological characteristics in the overall population. As expected, the expression of HAI-1 had a statistically significant association with clinicopathological parameters such as lymph node metastasis (p = 0.010) and LVS involvement (p = 0.005), but age, stage, grade, depth of myometrial invasion, cervical involvement, ovarian metastasis and peritoneal cytology were not considered to be statistically significantly associated. Likewise, the expression of HAI-2 was found to have a statistically significant association with clinicopathological parameters such as lymph node metastasis (p = 0.023) and LVS involvement (p = 0.003), but age, stage, grade, depth of myometrial invasion, cervical involvement, ovarian metastasis and peritoneal cytology were not statistically significant (Mann–Whitney U-test, p < 0.05).

Table 1. Association between HAI-1 and HAI-2 and clinicopathological factors in endometrial cancer
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The disease-free and overall survival analysis according to HAI-1 and HAI-2 expression status

HAI-1 and HAI-2 were most significantly exhibited on disease-free (DFS) and overall survival (OS) analysis of prognostic factors using the log-rank test for endometrial cancer. Figures 5a5f show the DFS and OS curves of 56 patients with endometrial cancer according to either HAI-1 or HAI-2, or both HAI-1 and HAI-2 expression status. The DFS and OS rates of patients exhibiting high HAI-1 or HAI-2, or HAI-1 and HAI-2 expression (score 2) were significantly higher than those of patients exhibiting low HAI-1 or HAI-2, or HAI-1 and HAI-2 expression (score 0–1) (DFS; p = 0.046, p = 0.026 and p = 0.019, OS; p = 0.050, p = 0.030 and p = 0.027, respectively) (Mann–Whitney U-test, p < 0.05). In particular, the DFS and OS curves of 56 patients exhibiting both high HAI-1 and HAI-2 expression were statistically significantly different.

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Figure 5. Kaplan–Meier plots for (a) disease-free and (b) overall survival of the 56 patients with endometrial cancer according to HAI-1 expression status. Kaplan–Meier plots for (c) disease-free and (d) overall survival of the 56 patients with endometrial cancer according to HAI-2 expression status. Kaplan–Meier plots for (e) disease-free and (f) overall survival of the 56 patients with endometrial cancer according to both HAI-1 and HAI-2 expression status. Low epithelial expression, score 0–1; high epithelial expression, score 2. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

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Discussion

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

HAI-1 and HAI-2 are Kunitz-type serine protease inhibitors that have a broad inhibitory spectrum against serine proteases. These serine protease inhibitors are type I transmembrane glycoproteins that contain two extracellular Kunitz-type inhibitory domains. This is the first study to investigate the role of the protease inhibitors of HAI-1 and HAI-2 in endometrial cancer.

These membrane-bound proteases are likely to have important roles in cellular homeostasis and their dysregulated activities and expression have been implicated in tumor development and progression. To date, several studies have suggested a possible role for HAI-1 and HAI-2 in various carcinoma cells. Our previous research also showed that HAI-1 and HAI-2 inhibited cell proliferation and invasion in cervical and ovarian cancers.11–13 In breast cancer, inactivation of HAI-1 and its homologous protein HAI-2 significantly increased HGF mediated breast cancer cell migration and invasion.41 Prostate cancer cells, after loss of HAI-1, showed an increase in invasiveness and cellular motility in vitro.42 Engineered overexpression of HAI-1 in glioblastoma cells reduced the invasiveness in vitro.43 We found that the potential inhibition by HAI-1 and HAI-2 mediated cell proliferation, migration and cellular invasion into endometrial cancer cells.

Inactivation of HAI-1 and HAI-2 might be promoted by multiple mechanisms. HAI-1 and HAI-2 are endogenous inhibitors of matriptase and hepsin. Both matriptase and hepsin are type II transmembrane proteins with an extracellular serine proteinase domain and show enhanced expression in a variety of tumor tissues. Therefore, the functional relevance of HAI-1 inhibition of matriptase was confirmed in a transgenic mouse model in which matriptase-induced skin tumorigenesis was completely prevented by the overexpression of HAI-1 in the same tissue.44 HAI-2 can also bind to high-affinity cell surface receptors and downregulate urokinase plasminogen activator (uPA) and its receptor (uPAR). Generation of plasmin from plasminogen by uPA can induce ECM degradation and promote tumor cell migration and metastasis. Matriptase and hepsin have been proposed to initiate signaling and proteolytic cascades through their ability to activate uPA and uPAR. Matriptase is known to interact with prostasin.45–49 Our previous research have assessed the level of expression of matriptase and hepsin during malignant progression and the potential value of matriptase and hepsin as a prognostic marker in endometrial cancer.20, 21 In this study, HAI-1 and HAI-2 were found to significantly inhibit matriptase and hepsin in endometrial cancer cells. Hence, proteinase activities have been implicated to function in EMT by activating growth factors and their receptors and cleaving cell–cell and cell–ECM adhesion molecules. The resultant signaling cascade induces E-cadherin transcription repressors such as Snail, Slug, SIP1 and Twist, eventually resulting in a loss of epithelial morphology.25–31 Cheng et al. have reported that HAI-1 has a significant regulatory role in EMT of the various carcinoma cells. The knockdown of HAI-1 reduced E-cadherin expression which led to loss of epithelial morphology and enhanced invasiveness in vitro. The effects of HAI-1 are likely mediated by reduction of matriptase, which results in an induction in the levels of E-cadherin and Slug, and a reduction in the levels of Vimentin, SIP1 and Snail.32 HAI-1 and HAI-2 caused a significant increase in expression of E-cadherin and Slug. On the other hand, the expression of Vimentin, SIP1, Snail and Twist were significantly decreased by HAI-1 and HAI-2.

Estrogen and progesterone pathways play an important role in the etiology of human endometrial carcinoma. The correlation of HAI-1 and HAI-2 with the activities of estrogen and progesterone pathways has not been studied. For this reason, experiments were conducted to determine the effects of HAI-1 and HAI-2 on the ERE and PRE. ER and PR signal transduction were significantly decreased by HAI-1 and HAI-2. Overall, the inhibition by HAI-1 and HAI-2 mediated cell proliferation, migration and cellular invasion by reduction of matriptase and hepsin expression, which in turn resulted in increasing the levels of E-cadherin and Slug, and reducing the levels of Vimentin, SIP1, Snail and Twist, and hence, ER and PR in vitro signal transduction in endometrial cancer.

It has been reported that reduced expression of HAI-1 is likely involved in the progression of prostate, breast and gastric cancers.14–16 In addition, the reduced expression of HAI-2 is possibly involved in disease progression in glioblastoma, hepatocellular carcinoma and renal carcinoma.17–19 Likewise, our previous reports indicated that reduced expression of HAI-1 and HAI-2 are possibly involved in disease progression in cervical and ovarian cancers.11–13 Szabo et al. have reported that the surface epithelium and uterine glands of the normal human uterus tract showed high expression levels of both HAI-1 and HAI-2.50 In the current study, the levels of HAI-1 and HAI-2 expression were significantly decreased in endometrial cancer specimens relative to corresponding normal endometrium specimens. We examined whether HAI-1 and HAI-2 expression was correlated with clinicopathological characteristics in patients with endometrial cancer. Both HAI-1 and HAI-2 expressions were found to have a statistically significant association with clinicopathological parameters such as lymph node metastasis and LVS involvement. Moreover, low levels of HAI-1 and HAI-2 expression were significantly associated with a poor prognosis for endometrial cancer patients. Interestingly, these findings indicate that HAI-1 and HAI-2 proteins could be important tumor suppressor genes for identifying uterine endometrium.

In summary, in vitro and in vivo studies have revealed a critical role for HAI-1 and HAI-2 in disruption of the basement membrane of endometrial cancer. These findings indicate that HAI-1 and HAI-2 may be possible tumor suppressor genes of endometrial cancer. HAI-1 and HAI-2 may be therapeutic targets for treatment and used as favorable prognosis markers for endometrial cancer.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

We are grateful to Dr. H. Kataoka for providing the full-length HAI-1 and HAI-2 cDNA to the Department of Section of Oncopathology and Regenerative Biology, Department of Pathology, Faculty of Medicine, University of Miyazaki, Japan. The ptk-ERE-Luc and ptk-PRE-Luc were kindly provided by Dr. S. Hayashi to the Division of Endocrinology, Saitama Cancer Center Research Institute, Japan.

References

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
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