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The Journal of Clinical Endocrinology & Metabolism Vol. 89, No. 11 5523-5534
Copyright © 2004 by The Endocrine Society

Nodal Induces Apoptosis and Inhibits Proliferation in Human Epithelial Ovarian Cancer Cells via Activin Receptor-Like Kinase 7

Guoxiong Xu, Yu Zhong, Sadia Munir, Burton B. Yang, Benjamin K. Tsang and Chun Peng

Department of Biology (G.X., Y.Z., S.M., C.P.), York University, Toronto, Ontario M3J 1P3, Canada; Sunnybrook and Women’s College Health Science Centre and Department of Laboratory Medicine and Pathobiology (B.B.Y.), University of Toronto, Toronto, Canada M4N 3M5; Department of Obstetrics and Gynaecology and Cellular and Molecular Medicine (B.K.T.), University of Ottawa, and Hormones, Growth and Development Program, Ottawa Health Research Institute, Ottawa, Canada K1Y 4E9

Address all correspondence and requests for reprints to: Dr. Chun Peng, Department of Biology, York University, 4700 Keel Street, Toronto, Ontario, Canada M3J 1P3. E-mail: cpeng{at}yorku.ca.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Human epithelial ovarian cancer is the most lethal female cancer. Hormones and growth factors, including the TGF-ß superfamily, have been suggested to play a role in ovarian tumorigenesis. The biological effects of TGF-ß superfamily are mediated by type I and type II serine/threonine kinase receptors and by intracellular Smad proteins. Recently, we have cloned four transcripts of human activin receptor-like kinase 7 (ALK7), a type I receptor for Nodal. In this study, we have investigated the role of Nodal and ALK7 in four ovarian cancer cell lines, OV2008, C13*, A2780-s, and A2780-cp. Overexpression of Nodal resulted in a significant decrease in the number of metabolically active cells. This effect was mimicked by a constitutively active ALK7 (ALK7-ca) but blocked by dominant negative mutants of ALK7, Smad2, or Smad3. Transient transfection of Nodal and ALK7-ca significantly decreased X-linked inhibitor of apoptosis protein (Xiap) expression, activated both caspase-3 and caspase-9, and increased apoptosis as determined by Hoechst nuclear staining and flow cytometry. In addition, Nodal and ALK7-ca also inhibited cell proliferation as measured by 5-bromo-2'-deoxyuridine (BrdU) assays. Interestingly, the effects of Nodal and ALK7-ca were more potent in chemosensitive A2780-s cells than in its chemoresistant counterpart, A2780-cp cells. These findings demonstrate that Nodal induces apoptosis and inhibits proliferation via ALK7 and Smad2/3 and that the effect of Nodal-ALK7 on apoptosis may be mediated in part by the down-regulation of Xiap and activation of caspase-9 and caspase-3.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE TGF-ß SUPERFAMILY has been shown to regulate a variety of cellular functions and to play an important role in tumorigenesis (1, 2, 3). This superfamily has over 35 members, including TGF-ßs, activins, bone morphogenetic proteins (BMPs), growth and differentiation factors, and other factors such as Nodal and its related proteins (1, 2, 3, 4, 5). Members of the TGF-ß superfamily signal through cell surface serine/threonine kinase receptors and intracellular Smad proteins (1, 2, 3, 4, 5, 6). Two distinct receptors, termed type I and type II receptors, are required to form functional receptor complexes (1, 2, 3, 4, 5, 6). Five type II and seven type I receptors, referred to as activin receptor-like kinase (ALK) 1–7, have been characterized in mammals. Both type I and type II receptors have a transmembrane domain, an extracellular ligand binding domain, and an intracellular serine/threonine kinase domain (1). In addition, type I receptors also have a segment of glycine and serine residues (GS box) preceding the kinase domain (1). Binding of ligands to their type II receptors recruits and phosphorylates type I receptors at their GS domain (7) and subsequently induces an intracellular downstream signaling cascade involving the Smad proteins and in turn specifies the nuclear transcriptional targets (1). To date, eight Smads are identified in vertebrates and classified into three subfamilies: receptor-regulated Smads (R-Smads) including Smad1, -2, -3, -5, and -8; common Smad (Smad4); and inhibitory Smads, including Smad6 and -7 (1, 2, 3). Smad2 and -3 are phosphorylated by TGF-ß and activin type I receptors, whereas Smad1, -5, and -8 are phosphorylated by BMP type I receptors (5, 6, 7, 8). Upon type I receptor phosphorylation and activation, R-Smads are subsequently phosphorylated and form complexes with Smad4 and, in turn, translocate into the nucleus (1, 2, 3).

ALK7 was initially cloned from the rat as an orphan receptor (9, 10) and has now been identified to be a type I receptor for Nodal (11). In a recent study, ALK7 was found to collaborate with activin receptor type IIB (ActRIIB) to mediate signaling by Nodal and its related proteins during embryonic development (11). Human ALK7 cDNA has been recently cloned (12, 13). We have also discovered that there are four transcripts of ALK7 generated from alternative splicing of the ALK7 gene (12). Three of the transcripts encode novel isoforms of ALK7, including a truncated receptor lacking a complete receptor binding domain, and two soluble proteins that have no transmembrane domain (12). The kinase domain of ALK7 is highly similar to that of ALK4 (activin receptor type IB) and ALK5 (TGF-ß receptor type I), and indeed it can induce the same R-Smads (Smad2 and Smad3) as ALK4 and ALK5 (13, 14, 15).

Nodal is an important regulator of embryonic development. It plays crucial roles in mesoderm formation and left-right axis patterning (16, 17, 18). During early embryogenesis, Nodal signals through ActRIIB and either ALK4 or ALK7 (11). In addition, Cripto, a protein belonging to the epidermal growth factor-Cripto, FRL-1, and Cryptic (EGF-CFC) family, has also been implicated in Nodal signaling. Cripto is essential for Nodal signaling through ALK4 (11, 19). It can also enhance the signaling of Nodal-ALK7 (11). Little is known about the role of Nodal in postnatal development. However, Nodal has been recently found to be expressed in mouse mammary gland (19), suggesting that Nodal may play a role in adult tissues.

Ovarian carcinoma is the most fatal gynecological malignancy and is the fourth leading cause of cancer in women (20, 21, 22). There are three major types of human ovarian cancers: epithelial, stromal, and germ cell (23). Among these, epithelial ovarian cancer (EOC), arisen from the ovarian surface epithelium, constitutes approximately 90% of cases of ovarian cancer (24). The most effective treatment in EOC is platinum-based chemotherapy, such as cisplatin. However, patients often develop chemoresistance and experience relapse (22).

OV2008 and A2780-s are two cisplatin-sensitive cell lines derived from two patients, whereas C13* and A2780-cp are their respective cisplatin-resistant variants established after in vitro cisplatin challenges (25, 26). These cell lines have been used for ovarian cancer research, including cell proliferation, apoptosis, chemotherapy, and chemoresistance (27, 28, 29, 30, 31). Using these cell lines, we have demonstrated that activation of the Nodal-ALK7 signaling pathway leads to induction of apoptosis and inhibition of cell proliferation. Furthermore, we have uncovered some of the mechanisms underlying the proapoptotic action of the Nodal-ALK7 pathway.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell lines and cell cultures

Human ovarian cancer cells OV2008, C13*, A2780-s, and A2780-cp cells were obtained and cultured as described previously (28, 29). Briefly, the cells were maintained in RPMI-1640 (OV2008 and C13*) or DMEM (A2780-s and A2780-cp) at 37 C in a humidified atmosphere of 5% CO2. The media were supplemented with 100 IU/ml penicillin and 100 µg/ml streptomycin in the presence of 10% fetal bovine serum. All culture media and reagents were purchased from Invitrogen Canada Inc. (Burlington, Ontario, Canada).

RNA extraction and RT-PCR

Total RNA was extracted from cells using TRIZOL reagent (Invitrogen) as described previously (32) and stored at –80 C until RT-PCR analyses. Two micrograms of total RNA were denatured at 70 C for 10 min in the presence of 0.2 µg random primers p(dN)6 (New England BioLabs Inc., Mississauga, Ontario, Canada) and then reverse transcribed in a final volume of 20 µl at 37 C for 2 h in 1x reaction buffer [50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol (DTT)] containing 200 U of Maloney murine leukemia virus reverse transcriptase (New England Biolabs), 0.5 mM dNTPs, 10 mM DTT, and 20 U RNase inhibitor (RNAguard, Pharmacia, Madison, WI). The reaction was terminated by heating the mixture at 95 C for 5 min. Amplification was carried out in a volume of 25 µl containing 1x reaction buffer (pH 8.7), 2 mM MgCl2, 0.2 mM dNTPs, 0.625 U HotStar Taq DNA polymerase (QIAGEN Inc., Mississauga, Ontario, Canada), and 10 pmol each of forward and reverse primers (synthesized by Sigma-Genosys, Oakville, Ontario, Canada). Primers for ALK7, Nodal, ActRIIB, Cripto, X-linked inhibitor of apoptosis protein (Xiap) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) are listed in Table 1Go. Primers for Smad2, -3, and -4 have been reported (31). Amplification was performed at 94 C for 30 sec, 54 C for 45 sec, and 72 C for 90 sec for 40 cycles [for ALK7 transcripts 1, 3, and 4 (ALK7-1, -3, and -4)]; at 94 C for 20 sec, 55 C for 30 sec, and 72 C for 45 sec for 22 (Smad2) and 25 (Smad3 and Smad4) cycles; or 94 C for 20 sec, 60 C for 30 sec, and 72 C for 45 sec for 18 (GAPDH), 25 (Xiap), 30 (ActRIIB, ALK7-1), 32 (Nodal), or 40 [ALK7 transcript 2 (ALK7-2) and Cripto)] cycles. For all PCRs, an initial step to activate HotStar Taq at 95 C for 15 min and a final extension of 10 min at 72 C were also performed. All primer sets used span at least one intron to eliminate the possibility of amplification from genomic DNA. In each PCR, possible contamination was checked by including a negative control in which water, instead of cDNA, was used as the template. PCR products were analyzed on a 1.5–2% agarose gel containing ethidium bromide.


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  		TABLE 1. Primers used for the detection of target mRNAs

 
Expression constructs

Expression constructs carrying the coding region of ALK7 or Nodal were made by cloning of ALK7 or Nodal into pEGFP-N1 (BD Biosciences Clontech, Mississauga, Ontario, Canada) and/or pcDNA4/TO/myc-His (Invitrogen). ALK7-1 and Nodal cDNAs were obtained by RT-PCR from the normal placental tissues using specific primers beginning with the start codon ATG and ending at the last codon just before the stop codon. The ALK7-1 primers with linkers of EcoRI and SacII were 5'-CGGAATTCCGCGATGACCCGGGCGCTCTGCT-3' (forward) and 5'-TCCCCGCGGGGCTTTGCAGTCTTCTTTGACACA-3' (reverse); the Nodal primers with an EcoRI linker on each end were 5'-CGGAATTCCACCATGCACGCCCACTGC-3' (forward) and 5'-CGGAATTCCGGAGGCACCCACATTCTTCCAC-3' (reverse). Wild-type ALK7 (ALK7-wt) with the ALK7-1 coding sequence was first cloned into the pEGFP-N1 and then subcloned into pcDNA4/TO/myc-His (version B) at HindIII and SacII sites. Nodal was directly cloned into the pcDNA4/TO/myc-His. The pcDNA4 plasmids were used in functional studies, whereas the pEGFP-ALK7-wt was used to determine transfection efficiency. Dominant negative Smad2 and Smad3 in pRK5F vector were obtained from Dr. Derynck (University of California at San Francisco, San Francisco, CA) and subcloned into pcDNA3.1 (Invitrogen) for these studies. All plasmids were fully sequenced using an ABI 373A sequencer at York University’s Core Molecular Biology Facility.

Site-directed mutagenesis

Constitutively active ALK7 (ALK7-ca) and kinase-defective ALK7 (ALK7-kd) were generated, based on the pcDNA4-ALK7-wt plasmid. Two mutagenic primers were designed to replace threonine-194 with aspartic acid (ALK7-ca) and lysine-222 with arginine (ALK7-kd). The oligonucleotide primers, each complementary to opposite strands of the vector, were extended during PCR by high-fidelity Taq DNA polymerase (Invitrogen). The product was then treated with DpnI to digest the parental DNA template, and the nicked vector DNA incorporating the desired mutations was transformed into DH5{alpha}. ALK7-ca and ALK7-kd were finally analyzed by restriction enzyme analysis and sequencing.

Transient transfection

Transient transfection was performed as described previously (33). Briefly, cells were plated and cultured for 24 h before transfection. After washing with OPTI-MEM I medium (Invitrogen), cells was transiently transfected using 25-kDa polyethylenimine (PEI, Sigma). One microgram of plasmid DNA required 0.28 µl of 0.18 mM PEI. First, the desired amount of plasmid DNA and the required PEI were diluted separately into 50 µl of 150 mM NaCl and incubated for 5 min. These solutions were then mixed and further incubated at room temperature for 12 min. The PEI/DNA mixture was then diluted into OPTI-MEM I medium and added to cells. After incubation for 5 h, culture medium was replaced with fetal bovine serum-containing medium and cells were recovered for different lengths of time as specified in each experiment. Transfection efficiency, estimated using pEGFP-ALK7-wt, was approximately 60% in 6-cm dishes and six-well plates and 40% in 96-well plates.

Luciferase reporter assay

A2780-cp cells were seeded into six-well plates at a density of 1.5 x 105 cells per well. After transient transfection of an empty vector pcDNA4 (EV), ALK7-wt, or ALK7-ca with p3TP-Lux (kindly provided by Dr. J. Massagué, Howard Hughes Medical Institute, New York, NY) and pRSV-ß-gal (obtained from Dr. A. Bédard, McMaster University, Hamilton, Ontario, Canada) overnight, cells were cultured in complete medium for 30 h. The luciferase activity was determined using a luciferase assay kit (Promega, Madison, WI) and measured using a luminometer. Data were normalized by the ß-gal activity.

Cell growth assay

Cell growth was measured by manual cell counting or by a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenly tetrazolium bromide (MTT) colorimetric method as reported earlier (34) with a cell proliferation kit I (Roche Diagnostics GmbH, Mannheim, Germany). For manual cell counting experiments, cells were plated into 6-cm dishes at 5 x 105 cells per dish for 24 h and then transiently transfected with an EV, ALK7-wt, or ALK7-ca for 5 h. Cells were then incubated in a complete medium for 24, 48, or 72 h, and cell numbers were then determined by hemocytometry. For MTT assays, cells were seeded into 96-well plates at a density of 104/100 µl per well and incubated for 24 h at 37 C before transfection. To test the dose-dependent effect of Nodal and ALK7-ca, cells were transiently transfected with an EV or varying amount of Nodal or ALK7-ca (0.1, 0.2, or 0.4 µg/well, and the total amount of DNA in each well was equalized by pcDNA4) for 5 h and then recovered in the complete medium for 72 h. In another experiment, cells were incubated with recombinant mouse Nodal (R&D Systems, Minneapolis, MN) at the concentration of 20, 100, and 500 ng/ml for 48 h. Cells were then incubated with 10 µl/well of the MTT solution during the last 4 h, followed by the addition of 100 µl solubilization buffer. After overnight incubation, the conversion of MTT to a colored product, formazan, by the intact mitochondria of living cells was determined by spectrophotometric measurement of absorbance at 595 nm. To determine the time course of Nodal and ALK7-ca action, after transfection with 0.2 µg/well of Nodal or ALK7-ca, cells were incubated in the complete medium for 24, 48, and 72 h with the MTT reagent added during the last 4 h of incubation. Finally, the combinational effect of Nodal and ALK7 was examined. Cells were transfected with 0.2 µg/well of EV, ALK7-ca, ALK7-kd, Nodal, dominant negative Smad2 (dS2), dominant negative Smad3 (dS3), Nodal plus dS2, Nodal plus dS3, Nodal plus ALK7-kd, ALK7-ca plus dS2, or ALK7-ca plus dS3 for 5 h; cells were recovered in the complete medium for 44 h and then incubated in MTT solution for 4 h. Each experiment was repeated at least twice.

Proliferation assay

Cell proliferation was determined by the measurement of BrdU incorporation in newly synthesized cellular DNA using a cell proliferation ELISA kit (Roche). Cells were seeded into 96-well plates at a density of 104/100 µl per well and incubated for 24 h at 37 C before transfection. Cells were then transfected with 0.2 µg/well of EV, Nodal, or ALK7-ca for 5 h. The assay was performed as described in the manufacturer’s instructions. Briefly, 10 µl/well of BrdU labeling solution was added to cells at 24, 48, or 72 h after transfection. After overnight incubation, cells were fixed with 100 µl/well of fix solution for 30 min at room temperature and incubated with anti-BrdU antibody conjugated with peroxidase for 90 min. A substrate solution was then added into each well, and absorbance was measured using an ELISA plate reader.

Assessment of apoptosis

Apoptotic cells were identified morphologically by Hoechst-33258 (bisBenzimide, Sigma) and 4',6'-diamidino-2-phenyindole (DAPI, Sigma) staining as previously reported (35) with slight modification. Briefly, cells were plated on 6-cm dishes at 5 x 105 cells/dish for 24 h, then transfected with an EV, Nodal, or ALK7-ca for 5 h. The cells were then incubated in a complete medium for 48 h. At the end of the culture period, suspended cells were collected by centrifugation and attached cells were trypsinized. The two cell fractions were then pooled, pelleted, and suspended in either 10% phosphate-buffered formalin containing 0.1 µg/ml Hoechst or 0.2 µg/ml DAPI in methanol. Cells were then incubated at room temperature for 20 min. After washing with PBS, cells were spotted on slides and assessed for typical apoptotic nuclear morphology (nuclear shrinkage, condensation, and fragmentation). To quantify the number of apoptotic cells, flow cytometry was performed. Again, cells were plated on 6-cm dishes and transfected with EV, Nodal, or ALK7-ca for 5 h. After recovery for 48 h, the cells were stained with Annexin V using a fluorescein isothiocyanate apoptosis detection kit I (BD Bioscience). Annexin V-positive cells were then measured using FACScan (Becton Dickinson, Mountain View, CA).

Western blot

Cell lysates were prepared from untransfected and EV-, Nodal-, or ALK7-ca-transfected cells. Equal amount of protein was separated by SDS-PAGE and transferred to a polyvinylidene difluoride membrane (Immobilon-P, Millipore Corp., Bedford, MA). After blocking with 5% milk in TBS-T, the membrane was incubated with mouse anti-c-myc (1:1000 dilution; Santa Cruz Biotechnology, Santa Cruz, CA), rabbit antihuman caspase-9 (1:1000 dilution; Santa Cruz), cleaved caspase-3 (Asp175) (1:1000 dilution, Cell Signaling, New England Biolabs Ltd., Pickering, Ontario, Canada), or Xiap (1:4000 dilution; Trevigen, Inc., Gaithersburg, MD) antibody at 4 C overnight or goat antihuman actin (1:1000 dilution; Santa Cruz) antibody at room temperature for 2 h. The membranes were subsequently probed with horseradish peroxidase-conjugated antimouse (Amersham, Baie d’Urfé, Québec, Canada), antirabbit (Amersham), or antigoat (Santa Cruz) secondary antibodies (1:5000 dilution) for 1 h. Signals were detected using ECL-Plus (Amersham).

Statistical analysis

Data from each experiment were expressed as percentage of the control value (EV), and in some cases, results from three to six experiments were pooled. Statistical analysis was done with one-way ANOVA, followed by a Tukey-Kramer multiple comparisons test (Graph Pad InStat Software, Graph Pad Inc., San Diego, CA). For comparison between two groups, Student’s t test was used. Differences were considered significant at values of P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Nodal and related signaling molecules are expressed in EOC cells

Expression of mRNAs for Nodal, ALK7, and related signaling molecules was determined by RT-PCR. Using primers that span exons III and IV, which are known to be alternatively spliced to generate different ALK7 transcripts (Fig. 1AGo), three DNA fragments corresponding to the expected sizes of ALK7-1, ALK7-3, and ALK7-4 were detected in OV2008, C13*, A2780-s, and A2780-cp cells (Fig. 1BGo). Expression of ALK7-2, Nodal, and ActRIIB, the type II receptor partner of ALK7, was also observed after RT-PCR with specific primers (Fig 1BGo). Furthermore, mRNA of Cripto, which has been shown to be a coreceptor for Nodal (11, 19), was expressed in these cells (Fig. 1BGo). Finally, Smad2, -3, and -4 mRNAs were also detected by RT-PCR in all four EOC cell lines (Fig 1BGo).



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  		FIG. 1. Detection of mRNAs of Nodal and its signaling components in OV2008, C13*, A2780-s and A2780-cp cells. A, Schematic presentation of the ALK7 gene and its transcripts. Transcript ALK7-1, translated into the full-length ALK7, is encoded by a gene consisting of nine exons. Transcript ALK7-3 lacks exon III, whereas ALK7-4 lacks exons III and IV; they encode proteins that have no transmembrane domain. Transcript ALK7-2 uses an alternative first exon (Ia), and the translated product has an incomplete receptor binding domain. UTR, Untranslated region; SP, signal peptide; RB, receptor binding domain; TM, transmembrane domain; GS, GS domain; Kinase, kinase domain. Locations of primers used in RT-PCR are indicated. B, RT-PCR for all ALK7 transcripts, Nodal, Cripto, ActRIIB, and Smad2, -3, and -4. Total RNA was extracted from ovarian cancer cells and reverse transcribed. RT-PCR was performed using specific primers (Table 1Go). A representative experiment is shown. RT-PCR for GAPDH (lower panels) was performed as an internal control.

 
Nodal, acting through ALK7 and Smad2/3, inhibits EOC cell growth

To assess the functionality of the ALK7 signaling cascade in EOC cells, a wild-type ALK7 expression plasmid and a constitutively active ALK7 construct were generated (Fig. 2AGo). A2780-cp cells were transiently cotransfected with p3TR-Lux, a Smad2/3-dependent reporter construct, and EV, ALK7-wt, or ALK7-ca. Overexpression of ALK7-ca significantly induced promoter activity of p3TR-Lux (Fig. 2BGo), indicating that the construct is functional and it can activate Smad2/3-mediated gene transcription. To evaluate the effect of ALK7 on cell growth, A2780-s cells were transiently transfected with EV, ALK7-wt, or ALK7-ca, and cell number was determined at 1, 2, and 3 d after transfection. Overexpression of ALK7-wt slightly reduced cell numbers at 48 and 72 h after transfection, whereas overexpression of ALK7-ca significantly inhibited cell growth (Fig. 2CGo). The inhibitory effect of ALK7-ca on cell growth was further examined in all four cell lines. Transient transfection of ALK7-ca into OV2008, C13*, A2780-s, and A2780-cp cells all resulted in a significant decrease in cell numbers at 72 h after transfection (Fig. 2DGo). ALK7-ca caused a 30–35% decrease in cell number in OV2008 and A2780-s cells, whereas in the chemoresistant C13* and A2780-cp cells, the cell number was decreased by only 20–25% after ALK7-ca transfection. However, the difference in response to ALK7-ca between the chemosensitive cells and their respective chemoresistant counterparts is not statistically significant.



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  		FIG. 2. Effect of ALK7-ca in ovarian cancer cells. A, Generation of ALK7-ca and ALK7-kd plasmids. Schematic representation of partial structure of pcDNA4-ALK7-wt plasmid is shown. The plasmid contains the cytomegalovirus promoter for ALK7 with the c-myc and His6 tags and the SV40 promoter for Zeocin resistance gene. Using site-directed mutagenesis, ALK7-ca was made by replacing the threonine-194 with the aspartic acid (T194D), and ALK7-kd was made by replacing the lysine-222 with the arginine (K222R). B, Transcriptional activity of ALK7-ca. A2780-cp cells were transiently transfected with 0.5 µg p3TP-Lux, 0.5 µg pRSV-ß-gal, and 2 µg EV pcDNA4 (EV), wild-type ALK7 (WT), or ALK7-ca (CA), and luciferase activity was measured at 30 h after transfection. C, Temporal changes in A2780-s cell number after transfection. Cell number was determined at 24, 48, and 72 h after transfection with EV, WT, or CA. D, Effect of ALK7 on OV2008, C13*, A2780-s, and A2780-cp cell growth. Cells were transiently transfected with EV, WT, or CA. Cell number was determined by hemocytometer counting at 72 h after transfection and normalized as percentage of control. Data represent means ± SEM (n = 3–6 experiments). *, P < 0.05 compared with EV control.

 
To further determine the growth-inhibitory effects of ALK7-ca and to test whether Nodal also has a similar effect on cell growth, concentration-dependent and time-course studies were performed using MTT assays to measure the number of metabolically active cells after Nodal and ALK7-ca transfection in A2780-s (Fig. 3AGo) and A2780-cp (Fig. 3BGo) cells. Nodal and ALK7-ca significantly decreased cell numbers in a concentration- and time-dependent manner in both cell types. However, A2780-S is more sensitive than A2780-cp cells, because a significant effect of Nodal and ALK7-ca was observed at 24 h after treatment in A2780-s cells, whereas a significant decrease in A2780-cp cell number was found only at 48 h after Nodal or ALK7-ca transfection. In addition, Nodal and ALK7-ca caused a maximum of 35% decrease in A2780-s cells (Fig. 3AGo), but the maximum response in A2780-cp cells was only 25% (Fig. 3BGo). Finally, we also used recombinant mouse Nodal to confirm the result obtained from Nodal overexpression studies. Cells were incubated with different amounts of recombinant mouse Nodal for 48 h, followed by MTT assays. As shown in Fig. 3CGo, Nodal induced a dose-dependent decrease in the number of metabolically active cells. Again, A2780-s cells are more sensitive to A2780-cp cells. First, at the dose of 100 ng/ml, Nodal caused a significant decrease in cell growth in A2780-s cells but not in A2780-cp cells. Second, the inhibition induced by Nodal in A2780-s cells (42%) was significantly greater than that in A2780-cp cells (27%).



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  		FIG. 3. Time- and dose-dependent effects of Nodal and ALK7-ca on cell growth/survival. A2780-s (A) and A2780-cp (B) cells were transiently transfected with EV, Nodal, or ALK7-ca plasmid DNA at different concentrations (0.1–0.4 µg/well). Cell growth was determined by the MTT assay at 72 h after transfection. For time-course studies, cells were transfected with 0.2 µg/well of Nodal or ALK7-ca plasmid DNA and cell growth determined by MTT assay at 24, 48, and 72 h after transfection. C, A2780 cells were incubated with different concentrations of recombinant mouse Nodal for 48 h, and cell growth/survival was measured by MTT assays. Data represent means ± SEM of six to eight wells of a representative experiment. *, P < 0.05 vs. the control; §, P < 0.05 vs. the corresponding treatment group in A2780-cp cells.

 
To confirm that the response observed after Nodal transfection was the result of an increase in Nodal expression, we first extracted total RNA from nontransfected cells or cells transfected with EV or Nodal- or ALK7-ca-transfected cells. The RNA samples were then treated with DNase to eliminate any genomic and plasmid DNA. RT-PCR with primers specific for Nodal revealed that Nodal mRNA levels were much higher in Nodal-transfected cells than that in the control or ALK7-ca-transfected cells. Similarly, using primers that are specific for ALK7 transcript 1 (Fig. 1AGo), an increased level of this transcript was observed in ALK7-ca-transfected cells (Fig. 4AGo). Western blot analysis with anti-c-Myc antibody was also performed using protein samples extracted from nontransfected cells and EV-, Nodal-, or ALK7-ca-transfected cells. Expected size bands corresponding to the propeptides of Nodal and ALK7 were observed in Nodal- and ALK7-ca-transfected samples. No signal was detected in control cells (Fig. 4BGo).



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  		FIG. 4. mRNA and protein expression of Nodal and ALK7-ca in transfected cells. Total RNA and protein were extracted from nontransfected cells (control) and EV-, Nodal-, or ALK7-ca-transfected cells at 48 h after transfection. A, Total RNA was treated with DNase before being reverse transcribed. PCRs were performed using primers specific for ALK7-1 and Nodal, respectively, for 30 (Nodal and ALK7-1) or 18 (GAPDH) cycles. B, Total protein was subjected to SDS-PAGE, followed by Western blot using an anti-c-myc antibody. Nodal and ALK7-ca containing c-myc tag were detected in Nodal and ALK7-ca transfected but not in nontransfected or EV-transfected cells. Equal protein loading was shown by Western blot for ß-actin.

 
To determine whether Nodal acts through ALK7 and Smad2/3 to inhibit cell growth, cells were transfected with Nodal alone or in combination with dominant negative mutants of ALK7 (ALK7-kd), Smad2 (DN-Smad2), or Smad3 (DN-Smad3). As shown in Fig. 5Go, overexpression of Nodal and ALK7-ca reduced the number of metabolically active cells significantly. However, when Nodal was cotransfected with ALK7-kd, DN-Smad2, or DN-Smad3, its inhibitory effect on cell growth was abolished. DN-Smad2 and DN-Smad3 also blocked the action of ALK7-ca.



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  		FIG. 5. Nodal acts through ALK7 and Smad2/3 to inhibit cell growth/survival. A2780-s (A) and A2780-cp (B) cells were transiently transfected with pcDNA4 (EV); Nodal (N); ALK7-ca (CA); ALK7-kd (KD); dominant negative Smad2 (dS2); dominant negative Smad3 (dS3); N plus KD, dS2, or dS3; or CA plus dS2 or dS3. The number of metabolically active cells was determined by MTT assays at 48 h after transfection. Data represent means ± SEM (n = 8 wells). *, P < 0.05 vs. EV control. The experiment was repeated twice with similar results.

 
Nodal and ALK7-ca inhibit EOC cell proliferation

Because the decrease in cell number can result from a decrease in cell proliferation and/or an increase in apoptosis, we then used BrdU assays to measure cell proliferation after Nodal and ALK7-ca transfection. A slight decrease in cell proliferation was observed at 24 h after Nodal or ALK7-ca transfection, whereas significant inhibition of cell proliferation was found at 48 and 72 h after Nodal or ALK7-ca transfection in both A2780-s and A2780-cp cells (Fig. 6Go).



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  		FIG. 6. Overexpression of Nodal and ALK7-ca inhibited cell proliferation as determined by BrdU assay. A2780-s and A2780-cp cells were transiently transfected with 0.2 µg of EV, Nodal (N), or ALK7-ca (CA) for 24, 48, or 72 h. Data represent means ± SEM (n = 8 wells). *, P < 0.05 compared with their respective controls.

 
Nodal and ALK7-ca induce EOC cell apoptosis by regulating Xiap and caspases

To determine whether the Nodal-ALK7 pathway also plays a role in regulating apoptosis, EOC cells were transiently transfected by Nodal, ALK7-ca, or their EV control. Typical morphological features of apoptotic nuclei, such as nuclear shrinkage, DNA condensation, and fragmentation, were observed in A2780-s and A2780-cp cells after Nodal and ALK7-ca transfection after either Hoechst staining (Fig. 7AGo) or DAPI staining (data not shown). When the number of apoptotic cells was analyzed by Annexin V labeling and flow cytometry, it was found that overexpression of Nodal and ALK7-ca increased the number of apoptotic cells (Fig. 7Go, B and C). In addition, ALK7-ca induced a significantly greater apoptosis in A2780-s cells than in its chemoresistant variant (Fig. 7CGo).



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  		FIG. 7. Induction of apoptosis by Nodal and ALK7-ca. A, Detection of apoptotic cells by Hoechst staining. Cells were transiently transfected with an EV, Nodal, or ALK7-ca. Apoptotic cells were observed in Nodal- and ALK7-ca-transfected cells. White arrows indicate the DNA condensation, shrinkage, and fragmentation. B, Typical flow cytometry profiles of Nodal- or ALK7-ca-transfected cells overlaid with the control profile (pcDNA4 transfected). C, Quantification of apoptosis in A2780-s and A2780-cp cells by flow cytometry. Data represent percentage of apoptotic cells (means ± SEM of three experiments). *, P < 0.05 vs. the EV control; §, P < 0.05 vs. the corresponding treatment group in A2780-cp cells.

 
Xiap is a key member of the inhibitor of apoptosis protein family, which blocks apoptosis by inhibiting the activation and activity of caspase-3 (35). Previous studies in EOC cells have demonstrated that Xiap plays a critical role in cisplatin-induced EOC cell apoptosis (36). To determine the mechanism involved in Nodal-ALK7-induced EOC cell apoptosis, we examined mRNA and protein levels of Xiap after Nodal and ALK7-ca transfection. As shown in Fig. 8AGo, Xiap mRNA levels decreased at 24 h after Nodal or ALK7-ca transfection when compared with levels found in EV-transfected cells and in nontransfected cells. The decrease in Xiap mRNA levels was observed from 12–48 h after transfection (data not shown). Similarly, Xiap protein levels were significantly down-regulated by the overexpression of Nodal and ALK7-ca at 48 h after transfection (Fig. 8BGo). Interestingly, Xiap levels in Nodal- and ALK7-ca-transfected A2780-s cells were also significantly lower than those in the A2780-cp cells, indicating that A2780-s cells have a stronger response to Nodal and ALK7-ca than A2780-cp cells (Fig. 8BGo). Because Xiap is known to inhibit caspase-9 and caspase-3 activities, we then determined whether there are changes in the activation of these caspases. In both A2780-s and A2780-cp cells, the amount of cleaved caspase-9 was increased by overexpression of Nodal and ALK7-ca (Fig. 8CGo). Similarly, the amount of active caspase-3 was much higher in Nodal- or ALK7-ca-overexpressing cells (Fig. 8CGo).



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  		FIG. 8. Effect of Nodal and ALK7-ca on expression of Xiap, caspase-9, and caspase-3 in A2780 cells. A, Nodal and ALK7-ca decreased Xiap mRNA levels. A representative RT-PCR experiment is shown. B, Nodal and ALK7-ca down-regulated Xiap protein content. A representative Western blot experiment as well as densitometry data from three experiments is shown. *, P < 0.05 vs. EV control; §, P < 0.05 vs. their corresponding treatment groups in A2780-cp cells. C, Western blots of caspase-9 and caspase-3. Total protein was subjected to SDS-PAGE. Nodal and ALK7-ca increased the cleaved active caspase-9 and caspase-3 protein content. ß-actin is used as control for the equal loading and normalization. Left and right panels are data from A2780-s and A2780-cp cells, respectively.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The present study demonstrated for the first time that Nodal signals through ALK7 and Smad2/3 to induce apoptosis and to inhibit proliferation in EOC cells.

Previously, we have shown that there are four ALK7 transcripts derived from alternative splicing of the ALK7 gene (12). Transcripts 1, 2, 3, and 4 encode, respectively, for a full-length receptor, a truncated receptor that is missing part of the ligand binding domain, and two soluble proteins that lack the transmembrane and GS domains (12). In this study, RT-PCR with primers specific for four transcripts of ALK7 generated DNA fragments of the expected sizes in OV2008, C13*, A2780-s, and A2780-cp cells, indicating that all ALK7 transcripts are expressed in these EOC cells. Based on our functional studies using both ALK7-ca and ALK7-kd constructs, it is clear that transcript 1 encodes for a functional receptor. The other transcripts of ALK7 are derived from alternative splicing and encode for ALK7 isoforms that have an incomplete receptor binding domain or have no transmembrane domain (12). Although the function of these ALK7 isoforms is not yet known, it is possible that they may modulate ALK7 signaling. The expression of ALK7 transcripts in ovarian cancer cells suggests that the ALK7 signaling pathway may play a role in regulating EOC cell activity.

ALK7 was initially identified as an orphan receptor because it was unable to bind TGF-ß, activin, or BMP7, even in the presence of their corresponding type II receptors TßRII, ActRIIA, and BMPRII (9, 10). Recent study has shown that ActRIIB and ALK7 can form a functional receptor complex for Nodal and Xenopus Nodal related factor 1 (Xnr1) (11). The constitutively active form of ALK7 mimicked the activity of Nodal in mesoderm induction, whereas a dominant negative ALK7 abolished the effect of Nodal (15), indicating that Nodal is a physiological ligand of ALK7. It has been found that, similar to ALK4 and ALK5, ALK7 also activates a signaling pathway involving Smad2 and -3 (15). Using RT-PCR, we observed that Nodal, Cripto, ActRIIB, and Smad2, -3 and -4 mRNAs are expressed in all four cell lines tested. This finding suggests that Nodal may play an autocrine regulatory role in these cells. Although Smads and ActRIIB have been found in some ovarian cancer cells (37, 38), to our knowledge, they have not been examined in OV2008, C13*, A2780-s, and A2780-cp cells. Recently, Adkins et al. (39) reported that Nodal mRNA is expressed in a number of cell lines, including OVCARs that are derived from ovarian carcinoma.

Only a few studies have examined the function of ALK7. In a rat pheochromocytoma cell line, overexpression of ALK7-ca has been found to inhibit cell proliferation and to induce cell differentiation (14). During the preparation of this manuscript, Kim et al. (40) reported that ALK7-ca stimulates apoptosis in hepatoma cells. In this study, we found that activation of ALK7 induced apoptosis and inhibited proliferation in OV2008, C13*, A2780-s, and A2780-cp cells. We also observed that overexpression of ALK7-wt slightly, but not significantly, decreased cell numbers. The small response observed after ALK7-wt transfection is likely because Nodal and the type II receptor partner, which are required for the activation of ALK7, were not overexpressed. Also, in our transient transfection system, only 40–60% of cells were transfected. A stronger effect of ALK7-wt would be expected with higher transfection efficiency. Nevertheless, the observation that ALK7-wt slightly decreased cell number, together with the expression of Nodal and ActRIIB in the ovarian cancer cell lines, suggest that the endogenous Nodal may activate ActRIIB, which in turn activates ALK7, resulting in a slight growth inhibition.

Nodal and its related proteins play important roles in mesoderm formation and left-right axis patterning during vertebrate development (16, 17, 18). It has been shown that overexpression of mouse Nodal and Xenopus Nodal related factors 1, 2, and 4 (Xnr1, 2, and 4) in embryo explants induces mesodermal tissue (41, 42, 43). Inhibition of Nodal signaling blocks mesoderm induction in Xenopus embryos (44) and prevents endoderm formation in the vegetal region (45). In this study, we found that overexpression of Nodal or incubation of EOC cells with mouse Nodal significantly decreased the number of metabolically active cells, demonstrating that Nodal inhibits EOC cell growth and/or survival. The effect of Nodal on EOC cell growth/survival could be blocked by dominant negative ALK7 and dominant negative Smad2 and Smad3, indicating that ALK7 and Smad2/3 mediate the actions of Nodal in ovarian cancer cells. These findings suggest that that the Nodal-ALK7 pathway can regulate cellular activities of ovarian cancer. The significance of Nodal-ALK7 signaling in ovarian tumorigenesis requires additional studies using normal and tumor samples derived from patients.

The decrease in cell number induced by Nodal or ALK7-ca overexpression is caused in part by an inhibition of cell proliferation as demonstrated by BrdU assays. Overexpression of Nodal and ALK7-ca led to significant decreases in BrdU incorporation into DNA, indicating that proliferation was inhibited by both Nodal and ALK7-ca. Preliminary studies in EOC cells have shown that Nodal and ALK7-ca up-regulate cell cycle inhibitors, such as p27 (Xu and Peng, unpublished data). Our recent studies in human trophoblast cells also demonstrate that activation of the Nodal-ALK pathway leads to an inhibition of cell cycle progression from G1 to S phase and this is caused in part by activation of p27 and inhibition of cdk2 (45). In PC12 cells, ALK7 was found to up-regulate p15 and p21, suggesting that these cell cycle inhibitors may be involved in ALK7-induced cell growth arrest (14). Additional studies are required to determine the mechanisms whereby Nodal-ALK7 inhibits EOC cell proliferation.

In morphological and flow cytometry studies, we demonstrated that the Nodal-ALK7 pathway induces apoptosis in EOC cells. Transient transfection of Nodal or ALK7-ca resulted in 4- to 6-fold increases in apoptotic cell numbers when compared with the control. To determine the mechanisms underlying the proapoptotic action of Nodal and ALK7, we measured Xiap mRNA and protein levels after Nodal and ALK7-ca transfection. Significant decrease in both Xiap mRNA and protein levels after Nodal and ALK7-ca transfection was found. Furthermore, caspase-3 and caspase-9 were activated by both Nodal and ALK7-ca. These results suggest that activation of the Nodal-ALK7 pathway down-regulates Xiap expression, which in turn activates caspase-9 and caspase-3, to induce apoptosis. However, Xiap may not be the only factor that is regulated by the Nodal-ALK7 pathway because a strong activation of caspase-3 and caspase-9 and only moderate decrease in Xiap expression was observed after ALK7 activation. It is well documented that caspase-3 and caspase-9 can be activated by the death receptor pathway through caspase-8 and by mitochondrial pathway through the release of a number of apoptogenic factors (46). In a rat hepatoma cell line, FaO, overexpression of ALK7-ca also resulted in activation of caspase-3 and caspase-9, but not caspase-8, suggesting that the death receptor pathway may not be involved in ALK7-induced apoptosis (40). Whether other signaling molecules upstream of caspase-9, and other members of the inhibitor of apoptosis protein (IAP) family, are involved in Nodal-ALK7 action in EOC cells will be investigated in the future.

Xiap is an important intracellular regulator of apoptosis, and its role in cancer formation, progression, and treatment has been suggested (36, 47). Levels of Xiap have been found to correlate with survival time; patients with lower Xiap levels had longer survival time than those with higher Xiap levels (35). Xiap has also been shown to play a direct role in the resistance of cancer cells, including EOC, to radiation and chemotherapies (36, 48). Thus, by regulating Xiap expression and apoptosis, the Nodal-ALK7 signaling pathway may play an important role in ovarian tumorigenesis.

Chemosensitivity is a major factor in determining the outcome of ovarian cancer treatment. Using both chemosensitive and chemoresistant EOC cell lines, previous studies have shown that Xiap, Akt2, and p53 are important mediators of chemoresistance (28, 36, 47, 48). In the present study, we used both chemosensitive and chemoresistant cells to determine the effect of Nodal-ALK7. Although Nodal and ALK7-ca inhibited proliferation and induced apoptosis in both cell lines, we consistently observed a slightly, and in several cases significantly, smaller response in A2780-cp cells than in A2780-s cells. In MTT assays, transient transfection with 0.1 µg of Nodal cDNA resulted in a significant decrease in cell growth/survival in A2780-s cells, but the same treatment had no effect on A2780-cp cells. Also, a significant decrease in cell growth/survival was observed in Nodal- or ALK7-ca-transfected A2780-s cells as early as 24 h after transfection, but no effect of Nodal or ALK7-ca was observed in A2780-cp cells until 48 h after transfection. Furthermore, recombinant mouse Nodal at 500 ng/ml induced more potent inhibition of cell growth/survival in A2780-s cells than in A2780-cp cells. In flow cytometry experiments, the number of apoptotic A2780-s cells induced by ALK7-ca was significantly higher than that of A2780-cp cells. Finally, there was a significant difference in the decrease in Xiap protein content after Nodal and ALK7-ca transfection between A2780-s and A2780-cp cells. These findings suggest that A2780-s cells are more sensitive to the activation of Nodal-ALK7 pathway than A2780-cp cells. It is not clear at present what factors contribute to the different sensitivity to Nodal-ALK7 between the chemosensitive and chemoresistant EOC cells. Because it is known that A2780-s cells have functional p53 whereas A2780-cp cells are p53 mutant (28), it is possible that the p53 pathway may be involved in Nodal/ALK7-regulated cell growth and apoptosis and, therefore, that the loss of p53 in A2780-cp cells causes a lower responsiveness to Nodal-ALK7. The precise role of the Nodal-ALK7 pathway in chemosensitivity of EOC cells remains to be investigated.

In summary, we have demonstrated that four EOC cell lines, OV2008, C13*, A2780-s, and A2780-cp, express the signaling molecules of the Nodal-ALK7 pathway, including the ligand, receptors, and Smads. Activation of the Nodal-ALK7 signaling pathway leads to EOC cell growth arrest. Moreover, Nodal-ALK7 signaling induces ovarian cancer cell apoptosis, and this effect is mediated, at least in part, by changes in the expression or activity of Xiap, caspase-9, and caspase-3. These novel findings suggest that the Nodal-ALK7 signaling pathway may play an important role in ovarian cancer development.


   Acknowledgments
 
We thank David laPierre for technical assistance in flow cytometry experiments.


   Footnotes
 
This work was supported in part by grants from the Canadian Institutes for Health Research (Grants MOP-53174 to C.P., MOP-62729 to B.B.Y., and MOP-15691 to B.K.T.) and National Cancer Institute of Canada (Grant 013335 to B.K.T.) and a Premier Research Excellent Award (to C.P.).

Abbreviations: ActRIIB, Activin receptor type IIB; ALK, activin receptor-like kinase; ALK7-1, ALK7 transcript 1; ALK7-2, ALK7 transcript 2; ALK7-3, ALK7 transcript 3; ALK7-4, ALK7 transcript 4; ALK7-ca, constitutively active ALK7; ALK7-kd, kinase-defective ALK7; ALK7-wt, wild-type ALK7; BMP, bone morphogenetic protein; BrdU, 5-bromo-2'-deoxyuridine; DAPI, 4',6'-diamidino-2-phenylindole; EOC, epithelial ovarian cancer; EV, empty vector; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide; PEI, polyethylenimine; R-Smad, receptor-regulated Smad; Xiap, X-linked inhibitor of apoptosis protein.

Received May 12, 2004.

Accepted July 21, 2004.


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 Discussion
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