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Cancer Research 67, 11377, December 1, 2007. doi: 10.1158/0008-5472.CAN-07-2803
© 2007 American Association for Cancer Research
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Experimental Therapeutics, Molecular Targets, and Chemical Biology

Down-regulation of Platelet-Derived Growth Factor-D Inhibits Cell Growth and Angiogenesis through Inactivation of Notch-1 and Nuclear Factor-{kappa}B Signaling

Zhiwei Wang1, Dejuan Kong1, Sanjeev Banerjee1, Yiwei Li1, N. Volkan Adsay2, James Abbruzzese3 and Fazlul H. Sarkar1

1 Department of Pathology, Karmanos Cancer Institute, Wayne State University, Detroit, Michigan; 2 Department of Pathology and Laboratory Medicine, Emory University School of Medicine, Atlanta, Georgia; and 3 Department of Gastrointestinal Medical Oncology, The University of Texas M. D. Anderson Cancer Center, Houston, Texas

Requests for reprints: Fazlul H. Sarkar, Department of Pathology, Karmanos Cancer Institute, Wayne State University School of Medicine, 9374 Scott Hall, 540 East Canfield, Detroit, MI 48201. Phone: 313-576-8327; Fax: 313-576-8389; E-mail: sarkarf{at}karmanos.org.


   Abstract
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 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Platelet-derived growth factor-D (PDGF-D) signaling plays critical roles in the pathogenesis and progression of human malignancies; however, the precise mechanism by which PDGF-D causes tumor cell invasion and angiogenesis remain unclear. Because Notch-1, nuclear factor-{kappa}B (NF-{kappa}B), vascular endothelial growth factor (VEGF), and matrix metalloproteinases (MMP) are critically involved in the processes of tumor cell invasion and metastasis, we investigated whether PDGF-D down-regulation could be mechanistically associated with the down-regulation of Notch-1, NF-{kappa}B, VEGF, and MMP-9, resulting in the inhibition of tumor cell invasion and angiogenesis. Our data showed that down-regulation of PDGF-D leads to the inactivation of Notch-1 and NF-{kappa}B DNA-binding activity and, in turn, down regulates the expression of its target genes, such as VEGF and MMP-9. We also found that the down-regulation of PDGF-D by small interfering RNA (siRNA) decreased tumor cell invasion, whereas PDGF-D overexpression by cDNA transfection led to increased cell invasion. Consistent with these results, we also found that the down-regulation of PDGF-D not only decreased MMP-9 mRNA and its protein expression but also inhibited the processing of pro-MMP-9 protein to its active form. Moreover, conditioned medium from PDGF-D siRNA–transfected cells showed reduced levels of VEGF and, in turn, inhibited the tube formation of human umbilical vascular endothelial cells, suggesting that down-regulation of PDGF-D leads to the inhibition of angiogenesis. Taken together, we conclude that the down-regulation of PDGF-D by novel approaches could lead to the down-regulation of Notch-1 and, in turn, inactivate NF-{kappa}B and its target genes (i.e., MMP-9 and VEGF), resulting in the inhibition of invasion and angiogenesis. [Cancer Res 2007;67(23):11377–85]


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Pancreatic cancer is one of the most aggressive cancers and is the fourth leading cause of cancer-related death in the United States (1). An estimated 37,000 new pancreatic cancer cases would be diagnosed and 33,370 deaths are expected in 2007 (1). This could be due to the fact that no effective methods of early diagnosis are currently available as well as due to the lack of effective systemic therapies resulting in the high mortality of patients diagnosed with pancreatic cancer. Presently, for all stages combined, the 1-year survival rate is only 20%, and the 5-year survival rate is <5% (1). This disappointing outcome strongly suggests that there is a dire need for designing new and targeted therapeutic strategies that could lead to a dramatic improvement in the survival of patients diagnosed with this deadly disease.

Pancreatic cancer, like many other tumors, has been shown to overexpress the platelet-derived growth factor (PDGF) family members (2–5). Four PDGF family members have been identified to date: PDGF-A, PDGF-B, PDGF-C, and PDGF-D. The PDGF-A, PDGF-B, and PDGF-C are secreted as homodimers or heterodimers and bind to dimeric PDGF receptors (PDGFR) composed of {alpha}- and/or β-chains, whereas PDGF-D can specifically bind to and activate PDGFRβ (2, 6–8). Since the 1970s, PDGF-A and PDGF-B have been extensively studied and well characterized, whereas PDGF-D was discovered only recently, and the functions of PDGF-D in human tumor progression especially in pancreatic cancer are largely unknown (7, 8).

It has been reported that PDGF-D signaling is frequently deregulated in human malignancies with up-regulated expression of PDGF-D in lung, prostate, renal, ovarian, and brain cancer (3, 9–13). These results suggest that PDGF-D plays important roles in the oncogenesis of several malignancies. Recent data suggest that overexpression of PDGF-D promoted tumor growth, angiogenesis, and metastasis of human renal cell carcinoma due to increased expression of angiopoietin-1 and matrix metalloproteinase-9 (MMP-9) in an orthotopic mouse model (9). Blocking PDGF-D/PDGFR signaling inhibited survival and mitogenic pathways in the glioblastoma cell lines and prevented glioma formation in a nude mouse xenograft model (12). There has been some progress toward elucidating the mechanism of action of PDGF-D as well as the consequence of down-regulation of PDGF-D; however, the exact mechanism has not yet been fully established. Therefore, we sought to find novel avenues by which PDGF-D could be inactivated, which may represent a promising strategy for the development of novel and selective anticancer therapies for pancreatic cancer. We investigated the consequence of down-regulation of PDGF-D by PDGF-D small interfering RNA (siRNA) on pancreatic cancer cell growth and apoptosis. Moreover, because cell migration and invasion are important processes involved in tumor development and metastasis and because PDGF-D signaling is known to control these processes, we also examined the effect of PDGF-D on the processes of cell migration and invasion of pancreatic cancer cells. We found that down-regulation of PDGF-D inhibits cell growth of pancreatic cancer cell lines. Our data also show that down-regulation of PDGF-D inhibited nuclear factor-{kappa}B (NF-{kappa}B) activity and the expression of Notch-1, MMP-9, and vascular endothelial growth factor (VEGF), which could be the mechanism responsible for the inhibition of pancreatic cancer cell migration, invasion, and the ability of conditioned medium to inhibit angiogenesis as measured by tube formation of human umbilical vascular endothelial cells (HUVEC). Collectively, our results suggest that down-regulation of PFGF-D by novel approaches could be useful strategy for the treatment of human pancreatic cancer.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture and experimental reagents. Human pancreatic cancer cell lines AsPC-1, BxPC-3, Colo-357, HPAC, L3.6pl, MIA PaCa, and PANC-1 were used in this study. BxPC-3, HPAC, and PANC-1 [American Type Culture Collection (ATCC)] were cultured in RPMI 1640 (Invitrogen) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin and streptomycin. AsPC-1, Colo-357, L3.6pl, and MIA PaCa cells were generously provided by Dr. Paul Chiao (M. D. Anderson Cancer Center, Houston, TX) and grown as a monolayer cell culture in DMEM containing 4.5 mg/mL D-glucose and L-glutamine supplemented with 10% FBS. HUVECs (ATCC) were cultured in F12K medium (ATCC) supplemented with 10% FBS, 0.1 mg/mL heparin sulfate, 0.05 mg/mL endothelial cell growth factor supplement (BD Biosciences), 100 units/mL penicillin, and 100 µg/mL streptomycin. All cells were cultured in a 5% CO2 humidified atmosphere at 37°C. Primary antibodies for Notch-1, cyclin D1, Bcl-2, MMP-9, and VEGF were purchased from Santa Cruz Biotechnology. Primary antibody for PDGF-D was obtained from R&D Systems. All secondary antibodies were obtained from Pierce. Lipofectamine 2000 was purchased from Invitrogen. Chemiluminescence detection of proteins was done with the use of a kit from Amersham Biosciences (Amersham Pharmacia Biotech). Protease inhibitor cocktail, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), and all other chemicals were obtained from Sigma.

Tissue material and immunohistochemistry. Tissue slides, including 20 human pancreatic adenocarcinoma sections (5 µm), were obtained from Karmanos Cancer Institute (Detroit, MI). The immunohistochemical determination of PDGF-D was accomplished as described earlier (14). Briefly, immunostaining was performed using PDGF-D antibody with appropriate dilutions and using normal host serum for negative controls followed by staining with appropriate horseradish peroxidase–conjugated secondary antibodies. The slides were developed in diaminobenzidine and counterstained with a weak solution of hematoxylin. The stained slides were dehydrated and mounted in Permount and visualized using an Olympus microscope. Images were captured with an attached camera linked to a computer.

Plasmids and transfections. PDGF-D siRNA and control siRNA were obtained from Santa Cruz Biotechnology. The PDGF-D cDNA plasmid was purchased from OriGene Technologies, Inc. Human pancreatic cancer cells were transfected with PDGF-D siRNA and cDNA, respectively, using Lipofectamine 2000 as described earlier (15).

Cell growth inhibition studies by MTT assay. The transfected cells (5 x 103) were seeded in a 96-well culture plate and subsequently incubated with MTT reagent (1.0 mg/mL) at 37°C for 2 h, and MTT assay was performed as described earlier (16). The results were plotted as mean ± SD of three separate experiments having six determinations per experiment for each experimental condition.

Western blot analysis. Cells were lysed in lysis buffer [50 mmol/L Tris (pH 7.5), 100 mmol/L NaCl, 1 mmol/L EDTA, 0.5% NP40, 0.5% Triton X-100, 2.5 mmol/L sodium orthovanadate, 10 µL/mL protease inhibitor cocktail, 1 mmol/L phenylmethylsulfonyl fluoride (PMSF)] by incubating for 20 min at 4°C. The protein concentration was determined using the Bio-Rad assay system. Total proteins were fractionated using SDS-PAGE and transferred onto nitrocellulose membrane for Western blotting as described earlier (15).

Real-time reverse transcription-PCR analysis for gene expression studies. The total RNA from transfected cells was isolated by Trizol (Invitrogen) and purified by RNeasy Mini kit and RNase-free DNase Set (Qiagen) according to the manufacturer's protocols. The primers used in the PCR for PDGF-D, PDGFRβ, Notch-1, MMP-9, and β-actin were described before (15, 17). Real-time PCR amplifications were performed as described earlier (15).

Electrophoretic mobility shift assay for measuring NF-{kappa}B activity. The transfected cells were washed with cold PBS and suspended in 0.15 mL of lysis buffer [10 mmol/L HEPES (pH 7.9), 10 mmol/L KCl, 0.1 mmol/L EDTA, 0.1 mmol/L EGTA, 1 mmol/L DTT, 1 mmol/L PMSF, 2 µg/mL leupeptin, 2 µg/mL aprotinin, 0.5 mg/mL benzamidine]. The nuclear protein was prepared and subjected to DNA-binding activity of NF-{kappa}B by electrophoretic mobility shift assay (EMSA) as described earlier (15).

Immunofluorescence staining. The cells were plated on coverslips in each well of an eight-well chamber for 24 h. Cells were then fixed with paraformaldehyde for 15 min, rinsed with PBS, and incubated with 5% goat serum for 30 min. The cells were then incubated with anti-Notch-1 antibody for 45 min. After washing with PBS, the cells were incubated with FITC-conjugated secondary antibody for 45 min and washed with PBS. Cell images were observed under a fluorescent microscope.

MMP-9 activity assay. The PDGF-D siRNA–transfected or PDGF-D cDNA–transfected cells were seeded in six-well plates and incubated at 37°C. After 24 h, the complete medium was removed and the cells were washed with serum-free medium. The cells were then incubated in serum-free medium for 24 h. MMP-9 activity in the medium was detected by using Fluorokine E Human MMP-9 Activity Assay kit (R&D Systems) according to the manufacturer's protocol.

VEGF assay. The PDGF-D siRNA–transfected or PDGF-D cDNA–transfected cells were seeded in six-well plates (1.0 x 105 per well) and incubated at 37°C. After 24 h, the cell culture supernatant was harvested and cell count was performed after trypsinization. After collection, the medium was spun at 800 x g for 3 min at 4°C to remove cell debris. The supernatant was either frozen at –20°C for later VEGF assay or assayed immediately using commercially available ELISA kits (R&D Systems).

Cell migration and invasion assay. Cell migration was assessed using 24-well inserts (BD Biosciences) with 8-µm pores according to the manufacturer's protocol. The invasive activity of the PDGF-D siRNA–transfected or control siRNA–transfected cells was tested using the BD BioCoat Tumor Invasion Assay System (BD Biosciences) as described earlier (18).

Matrigel in vitro HUVEC tube formation assay. The PDGF-D siRNA–transfected or PDGF-D cDNA–transfected cells were cultured in serum-free RPMI 1640 for 24 h. The conditioned media were collected, centrifuged, transferred to fresh tubes, and stored at –20°C. HUVECs were trypsinized and seeded (5 x 104 per well) in Matrigel-coated well with 250 µL of conditioned medium from PDGF-D cDNA–transfected or control plasmid–transfected BxPC-3 or MIA PaCa cells. The tube formation was assayed as described earlier (18).

Densitometric and statistical analysis. The cell growth inhibition after transfection was statistically evaluated using GraphPad StatMate software (GraphPad Software, Inc.). Comparisons were made between control and transfection. P < 0.05 was used to indicate statistical significance.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Overexpression of PDGF-D in human pancreatic cancer specimens. We first detected the PDGF-D expression levels in 20 human pancreatic tissue specimens. The results from the immunohistochemical staining showed that PDGF-D was expressed in the ducts of chronic pancreatitis specimens to a lesser degree (Fig. 1A ). However, PDGF-D was strongly expressed in the pancreatic adenocarcinomas (Fig. 1B and C). These results showed that most pancreatic adenocarcinomas express high levels of PDGF-D. Subsequently, we have scanned several human pancreatic adenocarcinoma cells for the expression of PDGF-D.


Figure 1
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  		Figure 1. Immunohistochemical expression of PDGF-D in pancreatic cancer tissue specimens. A, chronic pancreatitis showing expression of PDGF-D in reactive cells (pancreatitis) and islets but to a lesser degree in the normal ducts. B, left, adenocarcinoma: intercalated duct/centroacinar cells show strong labeling but the acinar cells are mostly negative; right, adenocarcinoma has moderate or strong degree of expression. C, adenocarcinoma showing moderate expression (high power).

 
Overexpression of PDGF-D in human pancreatic cancer cell lines. The baseline expression of PDGF-D was determined in a panel of human pancreatic cancer cell lines that included AsPC-1, BxPC-3, Colo-357, HPAC, L3.6pl, MIA PaCa, and PANC-1. All the pancreatic cancer cell lines are K-ras mutation positive except BxPC-3 cell lines. The results showed that PDGF-D was frequently but differentially expressed in different human pancreatic cancer cell lines (Fig. 2C ). We also examined the relative mRNA levels of PDGF-D in all seven pancreatic cancer cell lines by real-time reverse transcription-PCR (RT-PCR). All seven cell lines also expressed differential levels of PDGF-D mRNA (Fig. 2A). Because the cellular effects of PDGF-D are exerted through binding to PDGFRβ, we also detected the relative levels of PDGFRβ in both mRNA and protein levels by real-time RT-PCR and Western blotting, respectively. We found that all seven cell lines expressed differential levels of PDGFRβ mRNA (Fig. 2A). However, PDGFRβ protein levels could not be detected in any of these cell lines. The latter could be due to the lack of sensitivity of the commercial antibody used.


Figure 2
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  		Figure 2. PDGF-D expression in pancreatic cancer cell lines. CS, control siRNA; PS, PDGF-D siRNA; CP, control plasmid; PP, PDGF-D cDNA plasmid. A and B, PDGF-D (top) and PDGFRβ (bottom) mRNA levels were measured by real-time RT-PCR in seven pancreatic cancer cell lines and stable PDGF-D–transfected cell lines, respectively. C, PDGF-D protein level was measured by Western blotting in seven pancreatic cancer cell lines and PDGF-D–transfected cell lines. Columns, mean of three independent experiments; bars, SD. *, P < 0.05, relative to control.

 
Down-regulation of PDGF-D expression by siRNA inhibited cell growth and induced apoptosis. To determine whether PDGF-D could be an effective therapeutic target for pancreatic cancer, the effect of PDGF-D siRNA on cell growth of the pancreatic cancer cells was examined in BxPC-3, HPAC, and Colo-357 pancreatic cancer cells. The reason for choosing these three pancreatic cancer cell lines was due to the fact that these cell lines showed moderate or higher expression of PDGF-D. These three cell lines contain wild-type K-ras (BxPC-3) and mutation K-ras (HPAC and Colo-357). The efficacy of PDGF-D siRNA for knockdown of PDGF-D mRNA and protein was confirmed by real-time RT-PCR and Western blotting, respectively. We observed that both PDGF-D mRNA (data not shown) and protein levels (Fig. 2C) were barely detectable in PDGF-D siRNA–transfected cells compared with control siRNA–transfected cells. The cell viability was determined by MTT, and the effect of PDGF-D siRNA on the growth of cancer cells is shown in Fig. 3A . We found that down-regulation of PDGF-D expression caused cell growth inhibition in all three pancreatic cancer cell lines.


Figure 3
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  		Figure 3. Effects of altered PDGF-D expression on pancreatic cancer cell growth and apoptosis. A, inhibition of cancer cell growth by PDGF-D siRNA as measured by MTT assay. B, induction of cancer cell apoptotic death by PDGF-D siRNA as measured by ELISA. C, promotion of cancer cell growth by PDGF-D cDNA as measured by MTT assay. Points, mean of three separate experiments having six determinations per experiment for each experimental condition; bars, SD. *, P < 0.05; **, P < 0.01, relative to control.

 
To investigate whether the growth-inhibitory effects of PDGF-D siRNA are partially related to the induction of apoptosis, the effect of PDGF-D siRNA on apoptotic cell death was examined using an ELISA-based assay. These results provided convincing data that down-regulation of PDGF-D induces apoptosis in all three pancreatic cancer cell lines (Fig. 3B). These data suggest that the growth-inhibitory activity of PDGF-D down-regulation is partly attributed to an increase in cell death.

Overexpression of PDGF-D by cDNA transfection promoted cell growth and inhibited apoptosis. Pancreatic cancer cells BxPC-3, Colo-357, and MIA PaCa were transfected with human PDGF-D cDNA or empty vector alone. The reason for choosing these three pancreatic cancer cell lines was due to the fact that these cell lines showed moderate or lower expression of PDGF-D. The proteins were measured using Western blotting. The results showed that PDGF-D protein level was increased by PDGF-D cDNA transfection (Fig. 2C). The results also showed that PDGFRβ mRNA expression was increased in PDGF-D cDNA–transfected cells (Fig. 2B). PDGF-D cDNA–transfected cells showed significant promotion of cell growth compared with empty vector–transfected control cells (Fig. 3C). We also found that overexpression of PDGF-D protected cells from apoptosis to a certain degree (data not shown).

Down-regulation of PDGF-D decreased Notch-1 expression. It has been reported that Notch-1 is critically involved in the processes of tumor cell proliferation and apoptosis (19). PDGF-A has been shown to activate the expression of Notch-1 in certain cell lines (20). Therefore, we investigated whether Notch-1 was down-regulated by PDGF-D siRNA in pancreatic cancer cell lines. To explore whether PDGF-D siRNA transfection could decrease the expression of Notch-1, real-time RT-PCR and Western blotting were conducted. We found that both Notch-1 mRNA (data not shown) and protein levels (Fig. 4 ) were dramatically decreased in the PDGF-D siRNA–transfected cells. In addition, we found that the expression of Notch-1 downstream target genes, including Bcl-2 and cyclin D1, was also down-regulated in PDGF-D siRNA–transfected cells (Fig. 4). However, overexpression of PDGF-D by cDNA transfection led to an increase in the expression of Notch-1 and its target genes in BxPC-3 and MIA PaCa cells (Fig. 4). Next, we investigated whether PDGF-D cDNA transfection could lead to an increase in the activated Notch-1 in the nucleus of pancreatic cancer cells. Indeed, we observed higher level of Notch-1 protein in the nucleus in the PDGF-D cDNA–transfected cells (Fig. 4).


Figure 4
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  		Figure 4. Effects of altered PDGF-D expression on Notch-1 expression in human pancreatic cancer cells. A, the expression of Notch-1 and its target genes was detected by Western blotting. The expression of selected proteins was inhibited by PDGF-D siRNA and increased by PDGF-D cDNA transfection, respectively. B, the Notch-1 mRNA level was increased by PDGF-D cDNA transfection as measured by real-time RT-PCR. C, the PDGF-D cDNA–transfected cells were subjected to immunofluorescent staining using anti-Notch-1 antibody. Higher level of Notch-1 protein in the nucleus was found in the PDGF-D–transfected cells. DAPI, 4',6-diamidino-2-phenylindole.

 
Down-regulation of PDGF-D decreased NF-{kappa}B DNA-binding activity. PDGF has been reported to cross-talk with NF-{kappa}B signaling pathway (21). Therefore, we measured the NF-{kappa}B DNA-binding activity in PDGF-D–transfected cells. We found that down-regulation of PDGF-D by siRNA transfection decreased NF-{kappa}B DNA-binding activity (Fig. 5A ). However, PDGF-D overexpression by cDNA transfection significantly induced NF-{kappa}B DNA-binding activity in stably transfected cells compared with the control (Fig. 5A). The specificity of NF-{kappa}B DNA binding to the DNA consensus sequence was confirmed by supershift. The expression of MMP-9 and VEGF is regulated by NF-{kappa}B (22). We therefore investigated whether MMP-9 and VEGF were induced by PDGF-D cDNA transfection.


Figure 5
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  		Figure 5. A, nuclear proteins from siRNA- and cDNA-transfected cells were subjected to analysis for NF-{kappa}B DNA-binding activity as measured by EMSA. Left, down-regulation of PDGF-D inhibited NF-{kappa}B DNA-binding activity compared with control; middle, PDGF-D cDNA transfection caused activation of NF-{kappa}B DNA-binding activity in all three cell lines tested; right, NF-{kappa}B supershift analyses. EMSA experiments were done by additional 30-min incubations with polyclonal supershift antibodies against p65 before the addition of labeled probe. Lane 1, nonspecific antibody (anti-cyclin D1); lane 2, p65 antibody. B, left, Western blot analysis showed that PDGF-D cDNA transfection increased the expression of MMP-9 and VEGF; right, real-time RT-PCR showed that PDGF-D cDNA increased the expression of MMP-9 genes at mRNA level in pancreatic cancer cells. C, left, PDGF-D cDNA transfection increased the activity of MMP-9 in pancreatic cancer cells; right, PDGF-D cDNA transfection increased the secreted levels of VEGF in pancreatic cancer cells. *, P < 0.05; **, P < 0.01, relative to control.

 
Overexpression of PDGF-D increased MMP-9 gene transcription and their activities. It has been reported that MMP-9 expression is elevated in the stable PDGF-D–transfected renal carcinoma cell line (9). Therefore, we investigated whether MMP-9 was up-regulated by PDGF-D cDNA in pancreatic cancer cell lines. To explore whether PDGF-D cDNA transfection could increase the expression of MMP-9, real-time RT-PCR and Western blotting were conducted. We found that both MMP-9 mRNA and protein levels were dramatically increased in the PDGF-D cDNA–transfected cells (Fig. 5B). Next, we examined whether the overexpression of PDGF-D could lead to an increase in the MMP-9 activity in pancreatic cancer cells. We found a marked increase in the activity of MMP-9 in PDGF-D cDNA–transfected cells (Fig. 5B). However, down-regulation of PDGF-D by siRNA transfection led to a decrease in MMP-9 expression and activity in BxPC-3 and HPAC cells (data not shown).

PDGF-D cDNA increased VEGF expression and activity. It has been well documented that PDGF-D modulates VEGF expression in many tumor cell lines (7, 23). To further explore whether PDGF-D cDNA transfection could lead to an increase in VEGF expression and its biological activity, we examined the protein levels of VEGF and VEGF activity secreted in the culture medium. We found that overexpression of PDGF-D could lead to an increase in the protein levels and the amount of secreted VEGF (Fig. 5B and C). However, there was a marked decrease in the expression and the secretion of VEGF in PDGF-D siRNA–transfected BxPC-3 and HPAC cells (data not shown).

Overexpression of PDGF-D increased pancreatic cancer cell migration and invasion. MMP-9 and VEGF are critically involved in the processes of tumor cell migration, invasion, and metastasis. Because cell transfected with PDGF-D cDNA showed increased expression and activity of MMP-9 and VEGF, we tested the effects of PDGF-D overexpression on cancer cell migration and invasion. We found that overexpression of PDGF-D increased pancreatic cancer cell migration. Moreover, as illustrated in Fig. 6B , PDGF-D cDNA–transfected cells showed a high level of penetration through the Matrigel-coated membrane compared with the control cells. The value of fluorescence from the invaded pancreatic cancer cells was increased about 2- to 3-fold compared with that of control cells (Fig. 6B). However, PDGF-D siRNA–transfected BxPC-3 and HPAC cells showed a marked decrease in cell migration and invasion (data not shown).


Figure 6
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  		Figure 6. PDGF-D cDNA transfection increased pancreatic cancer cell migration and invasion and induced the HUVEC tube formation. *, P < 0.05, relative to control. A, top, migration assay showing that PDGF-D cDNA transfection increased pancreatic cancer cell migration; bottom, value of fluorescence from the migrated cells. B, top, invasion assay showing that PDGF-D cDNA transfection resulted in high penetration of cells through the Matrigel-coated membrane compared with control cells; bottom, value of fluorescence of the invaded cells. The values indicated the comparative levels of invaded cells. C, top, conditioned media from PDGF-D cDNA–transfected BxPC-3 and MIA PaCa cells were able to significantly induce the tube formation of HUVECs in 6-h incubation compared with the conditioned medium from control cells; bottom, image analysis of tubule/capillary length was carried out using software image analysis program Scion Image. Quantification of cumulative tube length of endothelial cells.

 
Increased tube formation of HUVECs induced by conditioned medium from PDGF-D cDNA–transfected cells. PDGF-D has been reported to promote both angiogenesis and metastasis in certain tumor models. Inhibition of PDGF-D reduced tumor cell proliferation and angiogenesis in human carcinomas (9, 23, 24). Because PDGF-D increased VEGF expression, we tested whether conditioned medium from PDGF-D cDNA–transfected cells could increase the tube formation, an indirect measure of angiogenesis. We performed the tube formation assay using HUVECs in growth factor–reduced Matrigel in vitro. As shown in Fig. 6C, conditioned media from PDGF-D cDNA–transfected BxPC-3 and MIA PaCa cells were able to significantly increase the tube formation of HUVECs in 6-h incubation compared with the medium from control plasmid–transfected cells. However, conditioned medium from PDGF-D siRNA–transfected BxPC-3 and HPAC cells showed reduced tube formation of HUVECs in 6-h incubation compared with the medium from control siRNA–transfected cells (data not shown).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
PDGF-D is a newly recognized growth factor, which can regulate many cellular processes, including cell proliferation, transformation, and migration, by activating its cognate receptor PDGFRβ (11, 23). It is known that PDGF-D interacts with PDGFRβ and activates downstream signaling, such as phosphatidylinositol 3-kinase (PI3K)/Akt, resulting in tumor development and progression. However, the precise role and mechanism of PDGF-D for tumor cell proliferation, invasion, and angiogenesis remains unclear. Here, we have provided molecular evidences showing that the down-regulation of PDGF-D could be an effective approach for the inactivation of Notch-1 and down-regulation of its target genes, such as NF-{kappa}B, MMP-9, and VEGF expression, resulting in the inhibition of invasion and angiogenesis of pancreatic cancer cells.

PDGF-D is important in the progression of several human cancers (9–13). However, the expression of PDGF-D and its role in human pancreatic cancer has not been previously investigated. Our study showed, for the first time, that PDGF-D is highly expressed in human pancreatic adenocarcinoma specimens, in chronic pancreatitis associated with pancreatic adenocarcinoma, and in five different human pancreatic cancer cell lines tested, suggesting that PDGF-D could be important in human pancreatic cancer progression. Because PDGF-D can specifically bind to and activate PDGFRβ, we also investigated whether PDGF-D signal plays a role through PDGFRβ. It has been reported that activated PDGFRβ was present in 90% of human pancreatic adenocarcinoma specimens (3). In addition, there was a 7-fold increase in the mRNA levels of PDGFRβ in the cancer samples by comparison with the normal pancreas (25). However, expression of PDGFRβ was barely detectable using Western blotting in pancreatic cancer cell lines (3, 25). Indeed, we found that all seven cell lines expressed differential levels of PDGFRβ mRNA. However, PDGFRβ protein could not be detected using Western blotting in any of these cell lines. We also found that PDGF-D cDNA transfection increased PDGFRβ mRNA level, suggesting that PDGF-D regulates the progression of pancreatic cancer through activation of PDGFRβ. It has been reported that LNCaP prostate cancer cells autoactivate PDGF-D, which can induce phosphorylation of PDGFRβ and stimulate cell proliferation in an autocrine manner. Additionally, LNCaP-processed PDGF-D, which functions in a paracrine manner (10, 11). Therefore, the activation of PDGFRβ by PDGF-D cDNA transfection in our system could be, in part, due to autocrine and/or paracrine manner.

It was reported that PDGF signaling regulates the expression of Notch-1 receptor in other cell lines (20). Notch-1 signaling is known to play important roles in maintaining the balance between cell proliferation, differentiation, and apoptosis (26). The Notch-1 gene is abnormally activated in many human malignancies, including pancreatic cancer (15, 19, 26). Notch-1 is known to play critical roles in the processes of tumor cell proliferation, invasion, and angiogenesis. In the present study, we found that down-regulation of PDGF-D inhibited the expression of Notch-1. Therefore, inactivation of PDGF-D–mediated cell invasion and angiogenesis could be partly mediated via inactivation of Notch-1 activity.

Previous studies have shown that Notch-1 activation could lead to the activation of NF-{kappa}B (15). NF-{kappa}B activation has also been reported to be associated with metastatic phenotype of tumor cells by regulating the expression of a variety of important genes known to be associated with many cellular responses (27). Because NF-{kappa}B plays important roles in many cellular processes, studies on the interaction of NF-{kappa}B activation with other cell signal transduction pathways, including the PDGF and Notch pathway, have received increased attention in recent years. PDGF has also been reported to cross-talk with the NF-{kappa}B pathway (21, 28). PDGF activates NF-{kappa}B through Ras and PI3K/Akt (21). In our early report, we showed that Notch-1 strongly induces NF-{kappa}B DNA-binding activity (15), which is consistent with previous findings from other laboratories (29, 30). In this study, we found that PDGF-D activates Notch-1 expression and consequently activated the DNA-binding activity of NF-{kappa}B. In addition, we also found that {gamma} secretase inhibitors, which inhibit Notch-1 activity, abrogated the PDGF-D–induced NF-{kappa}B DNA-binding activity (data not shown). Therefore, it is possible that PDGF-D–induced cell invasion and angiogenesis is partly due to activation of the NF-{kappa}B through Notch-1 activation.

It has been reported that PDGF-D promotes both angiogenesis and metastasis in certain tumor models and that inhibition of PDGF-D reduces tumor cell proliferation and metastasis in renal cell carcinomas (9). MMP-9 expression was also elevated in the PDGF-D–transfected renal carcinoma cell line (9). It is known that MMPs are critically involved in the processes of tumor cell invasion and metastasis and that MMP-9 is directly associated with angiogenesis and metastatic processes (31, 32). MMP-9 has been implicated in metastasis because of its role in the degradation of basement membrane collagen (31). Here, we showed that overexpression of PDGF-D increased MMP-9 expression. We also found that overexpression of PDGF-D increased the activity of MMP-9 in the culture medium of pancreatic cancer cells. However, down-regulation of PDGF-D inhibited the expression and activity of MMP-9. Thus, these results suggest that down-regulation of PDGF-D could potentiate antitumor and antimetastatic activities partly through the down-regulation of the expression of MMP-9.

Another important molecule involved in tumor cell invasion and angiogenesis is VEGF. Many studies have documented that VEGF is a critical mediator of angiogenesis and regulates most of the steps in the angiogenic cascade, including proliferation, migration, and tube formation of endothelial cells (33, 34). It has been reported that VEGF promotes migration and invasion of pancreatic cancer cells (34). Investigations by other laboratories have shown that PDGF modulates VEGF expression in many tumor cell lines, suggesting that PDGF-mediated signaling in tumors may accelerate both tumor cell growth and invasion of surrounding stroma, including stimulation of angiogenesis (7, 23). In this study, we found a marked increase in the secreted form of VEGF in PDGF-D cDNA–transfected cells. We also found a significant reduction of VEGF secretion in the culture medium of pancreatic cancer cells by down-regulation of PDGF-D using PDGF-D siRNA transfection.

Because we observed that overexpression of PDGF-D increased the expression and activities of MMP-9 and VEGF, we tested the effects of overexpression of PDGF-D on the migration and invasion of pancreatic cancer cells and tube formation (angiogenesis) of HUVECs. We found that overexpression of PDGF-D increased migration and invasion of pancreatic cancer cells through Matrigel and induced tube formation of HUVECs. These results are consistent with activation of MMP-9 and VEGF by overexpression of PDGF-D, resulting in the promotion of cancer cell invasion and angiogenesis. However, down-regulation of PDGF-D inhibited migration and invasion of pancreatic cancer cells through Matrigel and reduced tube formation of HUVECs. Based on our results, we speculate that one possible mechanism by which PDGF-D induces invasion and angiogenesis is by the activation of Notch-1 and NF-{kappa}B DNA-binding activity, which leads to up-regulation of NF-{kappa}B target genes, such as MMP-9 and VEGF. However, further in-depth studies are needed to ascertain the precise molecular regulation of PDGF-D and NF-{kappa}B and their cross-talks in elucidating the role of PDGF-D in cell growth, invasion, and angiogenesis of pancreatic cancer cells in animal models and in human pancreatic cancer.

In summary, we presented experimental evidence that strongly supports the role of PDGF-D down-regulation as antitumor and antimetastatic mechanisms in pancreatic cancer. Therefore, down-regulation of PDGF-D could potentially be an effective therapeutic approach for the inactivation of Notch-1 and NF-{kappa}B and its target genes, such as MMP-9 and VEGF, which is likely to result in the inhibition of cell growth, migration, invasion, angiogenesis, and metastasis of pancreatic cancer.


   Acknowledgments
 
Grant support: National Cancer Institute/NIH grant 5R01CA101870-05 (F.H. Sarkar), University of Texas M. D. Anderson Cancer Center Specialized Program of Research Excellence grant 1P20-CA010193-01 on pancreatic cancer (J. Abbruzzese), and Puschelberg Foundation.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   Footnotes
 
Note: Z. Wang and D. Kong contributed equally to this work.

Received 7/23/07. Revised 9/13/07. Accepted 10/ 5/07.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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