High expression of N-acetylglucosaminyltransferase IVa promotes invasion of choriocarcinoma

Gestational trophoblastic diseases (GTDs) are related to trophoblasts, and human chorionic gonadotropin (hCG) is secreted by GTDs as well as normal placentas. However, the asparagine-linked

sugar chains on hCG contain abnormal biantennary structures in invasive mole and choriocarcinoma, but not normal pregnancy or hydatidiform mole. N-acetylglucosaminyltransferase-IV (GnT-IV)

catalyses β1,4-N-acetylglucosamine branching on asparagine-linked oligosaccharides, which are consistent with the abnormal sugar chain structures on hCG.

We investigated GnT-IVa expression in GTDs and placentas by immunohistochemistry, western blot, and RT–PCR. We assessed the effects of GnT-IVa knockdown in choriocarcinoma cells in vitro and

in vivo.

The GnT-IVa was highly expressed in trophoblasts of invasive mole and choriocarcinoma, and moderately in extravillous trophoblasts during the first trimester, but not in hydatidiform mole or

other normal trophoblasts. The GnT-IVa knockdown in choriocarcinoma cells significantly reduced migration and invasive capacities, and suppressed cellular adhesion to extracellular matrix

proteins. The extent of β1,4-N-acetylglucosamine branching on β1 integrin was greatly reduced by GnT-IVa knockdown, although the expression of β1 integrin was not changed. In vivo studies

further demonstrated that GnT-IVa knockdown suppressed tumour engraftment and growth.

These findings suggest that GnT-IVa is involved in regulating invasion of choriocarcinoma through modifications of the oligosaccharide chains of β1 integrin.

Gestational trophoblastic diseases (GTDs) are a spectrum of cellular proliferations arising from placental villous trophoblasts and include hydatidiform mole and tumours that arise from

trophoblasts, such as invasive mole, choriocarcinoma, and placental site trophoblastic tumour (PSTT; World Health Organization., 1983). Hydatidiform moles are not tumours but abnormal

conceptuses caused by genetic fertilisation disorders, and are classified into two entities as follows: complete hydatidiform mole, which is androgenetic in origin, and partial hydatidiform

mole, which is mostly a dispermic triploid (Kajii and Ohama, 1977). Invasive mole is a benign tumour, which occurs after 10–20% of complete hydatidiform mole and 1–2% partial hydatidiform

mole (Goto et al, 1993). However, chemotherapy is needed for treatment of invasive mole because the tumour arises from myometrial invasion of molar villi, and 15–40% of patients show

metastasis to lung or vagina (Soper, 2006; Lurain, 2010). Choriocarcinoma is a malignant epithelial tumour that is associated with all kinds of pregnancies, but tends to develop from

hydatidiform mole more than normal delivery and abortion (Lurain, 2010). PSTT is a rare malignant tumour arising from intermediate trophoblasts in placental site (Shih and Kurman, 1998).

Although recently all invasive moles can be in primary remission, 1–3% of invasive moles have recurrence and the survival rate of choriocarcinoma and PSTT are about 85% and 70%, respectively

(Khan et al, 2003; Schmid et al, 2009). These indicates that invasive mole is a pre-malignant disease, and hydatidiform mole has a greater potential to develop into a malignancy than normal

pregnancy, but the mechanism by which trophoblasts become malignant remains unclear.

Human chorionic gonadotropin (hCG) is a glycoprotein hormone that is produced by syncytiotrophoblasts of human placentas as well as GTDs. Human chorionic gonadotropin is a heterodimer

composed of the αhCG and βhCG subunits. The αhCG subunit is asparagine-linked (N-linked) glycosylated at Asn-52 and Asn-78, and the βhCG subunit contains two N-linked sugar chains at Asn-13

and Asn-30, and four serine-linked (O-linked) sugar chains at Ser-121, Ser-127, Ser-132, and Ser-138 (Cole et al, 1984; Kobata and Takeuchi, 1999). The N-linked sugar chains of hCG in normal

pregnancy and hydatidiform mole contained monoantennary, biantennary, and fucosylated biantennary, but abnormal biantennary sugar chains were added in choriocarcinoma and a triantennary

sugar chain added in choriocarcinoma and invasive mole (Mizuochi et al, 1983; Endo et al, 1987).

N-acetylglucosaminyltransferase IV (GnT-IV), which transfers an N-acetylglucosamine (GlcNAc) group to the core α1,3mannose of N-glycans forming a β1-4 linkage, can act on biantennary sugar

chains and generate the triantennary sugar chains on hCG in invasive mole and choriocarcinoma. The activities and mRNA expression levels of glycosyltransferases (GnT-I to -V,

β1-4galactosyltransferase, and α-mannosidase II) were examined in placentas and choriocarcinoma, and the GnT-IV activities and the GnT-IVa mRNA level in the choriocarcinoma cell lines were

significantly higher than in the normal placentas (Takamatsu et al, 1999, 2004). However, no study has examined GnT-IVa protein expression and localisation in GTDs compared with normal human

placentas or the function of GnT-IVa in trophoblasts.

In this study, we examined GnT-IVa expression in trophoblastic cells of GTDs and normal placentas, and the role of GnT-IVa in trophoblastic cells, especially in choriocarcinoma.

Informed consent was obtained from patients for the use of placental samples and GTD tissue specimens. First-trimester and early second-trimester placentas were obtained from women

undergoing elective pregnancy terminations. Full-term placental samples were collected during elective Caesarean sections before the onset of labour. The GTD tissues were obtained from

patients who underwent surgical treatment, and the specimens were classified based on their histopathological characteristics. None of the patients had received chemotherapy for the disease

before surgery. All tissue samples were fixed in 10% formaldehyde, embedded in paraffin, and routinely stained with haematoxylin and eosin for histological examination. Some of the tissue

samples were washed with phosphate-buffered saline (PBS), frozen in liquid nitrogen immediately after removal, and then stored at −80 °C until protein extraction. This study was approved by

the ethics committee of Nagoya University Graduate School of Medicine.

Immunohistochemical staining was performed using the avidin−biotin immunoperoxidase technique. Sections (4-μm-thick) were immunostained as previously described (Yamamoto et al, 2005), using

an anti-GnT-IVa Ab (M-71; Santa Cruz Biotechnology, Santa Cruz, CA, USA) diluted 1 : 400 and anti-hCG Ab (N1534; DAKO, Carpinteria, CA, USA). To evaluate the expression level of GnT-IVa in

GTDs, the specimens of hydatidiform mole (n=11), invasive mole (n=4), choriocarcinoma (n=8), and PSTT (n=3) were used. The GnT-IVa expression levels were classified semiquantitatively, based

on the total scores of the per cent positivity of stained tumour cells and the staining intensity. Namely, the per cent positivity was scored as 0 if 70% of cells stained.

Human choriocarcinoma cell lines (Jar, BeWo, and JEG-3) were purchased from the American Type Culture Collection (Manassas, VA, USA). The NaUCC (CC)-1, CC-3, CC-4, and CC-6 are human

choriocarcinoma cell lines that were previously established in our laboratory (Ino et al, 1991). The human extravillous trophoblast (EVT) cell line HTR-8/SVneo was kindly provided by Dr

Charles H Graham (Graham et al, 1993). All cell lines were grown in RPMI 1640 (Sigma, St Louis, MO, USA), supplemented with 10% FCS, penicillin (100 U ml−1), streptomycin (100 μg ml−1), and

2 mM glutamine. Cultures were incubated at 37 °C in 5% CO2.

Western blot analysis for GnT-IVa protein was performed as previously described (Yamamoto et al, 2007), with an anti-GnT-IVa mAb (M-71; Santa Cruz Biotechnology) diluted 1 : 1000.

Immunoreactive proteins were stained using a chemiluminescence detection system (ECL; Amersham, Arlington Heights, IL, USA). An Ab against β-actin (AC-15; Sigma) was used to standardise the

protein loading.

Total RNA extraction and quantitative real-time PCR were performed as previously described (Mano et al, 2009). We used the following primers for GnT-IVa: forward primer,

5′-ACCAAGGGCATACGCTGGAG-3′; reverse primer, 5′-GTTCTTGGTTGCCGCTATGGA-3′, and the following primers for GAPDH: forward primer, 5′-CGGGAAACTGTGGCGTGAT-3′; reverse primer,

5′-ATGCCAGTGAGCTTCCCGT-3'. The PCR profile was an initial incubation at 95 °C for 10 s, followed by 45 cycles of denaturation at 95 °C for 5 s, and annealing and extension at 60 °C for 30 s.

Lectin blot analysis was performed as previously reported (Yamamoto et al, 2009), using HRP-labelled Datura stramonium agglutinin (DSA; Seikagaku, Tokyo, Japan) diluted 1 : 2000, and DSA

recognises β1-4GlcNAc branching.

Small interfering RNAs (siRNAs) were designed and synthesised by Nippon EGT (Toyama, Japan) to target GnT-IVa (siRNA1: 5′-AGAUGGCUAUUUCAGAAUATT-3′ and 5′-UAUUUCUGAAAUAGCCAUCUTT-3′; siRNA2:

5′-GAAGAUGGCUAUUUCAGAATT-3′ and 5′-UUCUGAAAUAGCCAUCUUCTT-3′). The non-targeting siRNAs (Nippon EGT) were used as a control (control siRNA: 5′-GGAUUAUUACGCAGUUAAATT-3′ and

5′-UUUAACUGCGUAAUAAUCCTT-3′). Jar cells were grown in 60-mm plates to 60% confluency and then transfected with 60 pmol of siRNAs using Lipofectamine RNAiMAX transfection reagent (Invitrogen,

Carlsbad, CA, USA) according to the manufacturer’s instructions. After treating Jar cells with siRNAs (siRNA1, siRNA2, and control siRNA) for 8 h, the medium containing the siRNAs and

transfection reagents were removed and cells were cultured in fresh culture medium for at least 24 h before each experiment. Transfected cells were cultured for 24 h before adhesion assay

and immunoprecipitation, and for 48 h before zymography and hCG assay.

To produce stable GnT-IVa knockdown cells for in vivo studies, the oligonucleotide sequences designed by Block-it RNAi Designer (Invitrogen) in the construction of the short hairpin RNA

(shRNA) vector were as follows: 5′-CACCGCTATTGTATGAGTCATAATTCGAAAATTATGACTCATACAATAGC-3′ and 5′-AAAAGVTATTGTATGAGTCATAATTTTCGAATTATGACTGATACAATAGC-3′. The shRNA vector was synthesised by

Invitrogen using the oligonucleotides and pENTR/H1/TO (Invitrogen). The vector plasmid was transfected into Jar cells using Lipofectamine 2000 reagent (Invitrogen) and selected by adding

zeosin. The original pENTR/H1/TO vector was used as a control shRNA.

Cells (5 × 103) were plated in 100 μl of medium in 96-well plates and incubated for 72 h at 37 °C. Cell viability was determined using the modified tetrazolium salt assay using the Cell

Titer 96 Aqueous One Solution Proliferation Assay kit (Promega, Madison, WI, USA) according to the manufacturer’s instructions. The mean values of three independent experiments performed in

eight wells are shown.

The migration and invasion assay was performed as previously reported (Yamamoto et al, 2005). Jar cells were transfected with siRNA 24 h before seeding for the migration and invasion assays,

and both assays were performed after 24 h incubation. The number of cells was counted under a microscope at × 200 magnification. Data were obtained from three individual experiments

performed in triplicate.

Cells (1 × 105) were plated in a 24-well chamber and incubated with serum-free medium for 48 h after siRNA transfection. Zymography was performed as previously reported (Yamamoto et al,

2009).

Cells (4 × 104) were plated in 96-well plates coated with fibronectin, collagen type I or type IV (Becton, Dickinson and Company, Franklin Lakes, NJ, USA), centrifuged at 1500 r.p.m. for 15 

s, and allowed to attach to each matrix at 37 °C for 30 min. After washing with PBS, absorbance readings at 492 nm (A492 nm) were performed using a microplate reader (Multiskan Bichromatic;

Labsystems, Helsinki, Finland). The rate of cell adherence was calculated as follows: (A492 nm (matrix)−A492 nm (no matrix))/A492 nm (no matrix) (Inamori et al, 2006; Yamamoto et al, 2009).

Data were obtained from three individual experiments performed in eight wells.

Immunoprecipitation was performed using 1.5 mg of protein that was extracted from cells with an anti-human βhCG mAb (HCG-60; Santa Cruz Biotechnology) or β1 integrin mAb (BV7; Abcam,

Cambridge, UK), as previously described (Yamamoto et al, 2007). The anti-βhCG mAb and anti-β1 integrin mAb (MAB2247; Chemicon International, Temecula, CA, USA) were used at a dilution of 1 :

 200 and 1 : 1000, respectively, and western blotting and DSA lectin blotting were performed using the above protocol.

Hormone assays were performed on culture supernatant. Cells (5 × 104) were plated in a 24-well chamber and incubated with 1 ml serum-free medium for 48 h after siRNA transfection. The total

hCG levels were quantified in triplicate by enzyme immunoassay using an αhCG mAb and a βhCG-CTP mAb (SRL Inc., Tokyo, Japan).

Female BALB/c slc nu/nu mice (5 weeks old) were purchased from Japan SLC (Nagoya, Japan).The treatment protocol followed the guidelines for animal experimentation adopted by Nagoya

University. Cells (5 × 106) per 0.2 ml of PBS/mouse were injected subcutaneously on the right flank to examine implantation and survival analysis by GnT-IVa stable knockdown. Each group

consisted of seven mice. The overall survival was defined as the time between the date of inoculation and the date of death due to tumour.

The non-parametric Kruskal–Wallis test was performed to compare immunostaining scores among all histological types. Data are expressed as the mean±s.d. For data of in vitro experiments,

statistical comparisons among groups were performed using the one-way ANOVA with Bonferroni corrections. Overall survival curves were analysed by the log-rank test. Differences were

considered significant when P