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Published as doi: 10.1096/fj.06-6138fje.
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(The FASEB Journal. 2006;20:1886-1888.)
© 2006 FASEB

Cellular prion protein promotes invasion and metastasis of gastric cancer

Yanglin Pan1, Lina Zhao1, Jie Liang, Jie Liu, Yongquan Shi, Na Liu, Guoyun Zhang, Haifeng Jin, Juan Gao, Huahong Xie, Jun Wang, Zhiguo Liu and Daiming Fan2

State Key Laboratory of Cancer Biology and Institute of Digestive Diseases, Xijing Hospital, the Fourth Military Medical University, Xi’an, Shaanxi Province, P. R. China

2Correspondence: State Key Laboratory of Cancer Biology and Institute of Digestive Diseases, Xijing Hospital, the Fourth Military Medical University, 17 Changle Western Rd., Xi’an, Shaanxi Province, 710032, P. R. China. E-mail: fandaim{at}fmmu.edu.cn

ABSTRACT

Cellular prion protein (PrPc) is a glycosylphosphatidylinositol (GPI) -anchored membrane protein that is highly conserved in mammalian species. PrPc has the characteristics of adhesive molecules and is thought to play a role in cell adhesion and membrane signaling. Here we investigated the possible role of PrPc in the process of invasiveness and metastasis in gastric cancers. PrPc was found to be highly expressed in metastatic gastric cancers compared to nonmetastatic ones by immunohistochemical staining. PrPc significantly promoted the adhesive, invasive, and in vivo metastatic abilities of gastric cancer cell lines SGC7901 and MKN45. PrPc also increased promoter activity and the expression of MMP11 by activating phosphorylated ErK1/2 in gastric cancer cells. MEK inhibitor PD98059 and MMP11 antibody (Ab) significantly inhibited in vitro invasive and in vivo metastatic abilities induced by PrPc. N-terminal fragment (amino acid 24–90) was suggested to be an indispensable region for signal transduction and invasion-promoting function of PrPc. Taken together, the present work revealed a novel function of PrPc that the existence of N-terminal region of PrPc could promote the invasive and metastatic abilities of gastric cancer cells at least partially through activation of MEK/ERK pathway and consequent transactivation of MMP11.—Pan, Y., Zhao, L., Liang, J., Liu, J., Shi, Y., Liu, N., Zhang, G., Jin, H., Gao, J., Xie, H., Wang, J., Liu, Z., Fan, D. Cellular prion protein promotes invasion and metastasis of gastric cancer.


Key Words: PrPc • extracellular matrix

THE NORMAL CELLULAR prion protein, designated PrPc, is a glycosylphosphatidylinositol (GPI) -anchored membrane protein that is highly conserved in mammalian species (1) . PrPc is expressed most abundantly in the brain, but has also been detected in other non-neuronal tissues such as muscle, lymphoid tissues and skin (2 3 4) . Although PrPc was negatively or weakly expressed in the neck region of the gastric mucosa, it was found to be up-regulated in the mucosa of patients with Helicobacter pylori infection (5) . Our previous data have demonstrated that PrPc was overexpressed in gastric cancer tissues, indicating a possible role for PrPc in occurrence and development of gastric cancer (6) .

Many investigations have focused on PrPc functions concerning physiological and pathological processes (7) . However, despite abundant information about the function of misfolding prion protein PrPsc, relatively little is known about the function of PrPc. Perhaps a well-studied aspect of PrPc is its ability to selectively bind copper ions through its octarepeat domain in N-terminal region. This binding confers redox property to PrPc (8) . Another emerging function of PrPc is its protective role in cell survival with regard to protection against oxidative stress, serum deprivation, and TNF{alpha}-induced apoptosis, especially in neuronal cells (9 10 11 12 13 14 15) . Our previous work demonstrated that PrPc could confer multidrug resistance to gastric cancer partially by inhibiting apoptosis of gastric cancer cells (6) . PrPc can also interact with DNA and RNA (16) . Recently, PrPc was shown to be involved in self-renewal of hematopoietic stem cells (17) .

PrPc is targeted to lipid-enriched microdomain, the so-called lipid raft, of the plasma membrane—regions that are abundant in receptors and signaling molecules—consistent with a role of PrPc in cell adhesion and membrane signaling. It has been shown that PrPc binds some extracellular matrix (ECM) and adhesive proteins, such as some glycosaminoglycans, laminin, N-CAM, laminin receptor, and laminin receptor precursor (18 19 20 21) . Moreover, PrPc can interact with itself through its octarepeat and C-terminal structured domains, and thus forms a homologous dimer (22) . The adhesive feature of PrPc is assumed to reflect some of its roles in cell adhesion, neurite growth, or even cell survival. It is indeed demonstrated that overexpression of PrPc induced cell adhesion and aggregation in neuroblastoma cells (23) . It is well known that adhesive molecules play important roles in the process of tumor metastasis, including adhesion, invasion, and survival in distant organs of tumor cells (24) . So we asked whether PrPc might influence invasive and metastatic abilities of gastric cancer cells.

Here we presented the first evidence that PrPc was highly expressed in metastatic gastric cancers. The existence of PrPc in gastric cancer could promote invasive and metastatic abilities of gastric cancer cells through activation of the MEK/ERK pathway and consequent transactivation of MMP11. Moreover, the N-terminal fragment was suggested to be an indispensable domain in transducing invasion-promoting signal of PrPc.

MATERIALS AND METHODS

Tissue collection and immunohistochemistry
Primary site tissues from 86 nonmetastatic gastric cancers and 38 metastatic gastric cancers, respectively, and tissues of primary and metastatic sites in lymph node from another 22 metastatic gastric cancers were obtained from patients who underwent surgery at the Department of General Surgery in Xijing hospital, Xi’an, China. Patients having surgical tissues dissected for the study signed informed consent. All cases of gastric cancer were clinically and pathologically proved. The protocols used in the study were approved by the Hospital’s Protection of Human Subjects Committee. Sections (5 µm) of formalin-fixed paraffin-embedded specimens were made. Slides were dewaxed, rehydrated, incubated in 10% normal goat serum and 0.3% Triton X-100 in PBS for 1 h, then incubated with monoclonal anti-PrPc Ab 3F4 (1:100, Sigma, Swampscott, MA, USA). The slides were washed in PBS three times for 5 min each. The tissues were incubated in biotin-labeled rabbit antimouse serum (1:200) for 30 min, rinsed with PBS, and incubated with avidin-biotin-peroxidase complex for 1 h. The signal was detected using 3,3-diaminobenzidine as the chromogen. Negative control slides omitting the primary Ab were included in all assays. Result was evaluated by formula as described previously (25) : the staining score = the intensity of immunoreactivity (IR) x the proportion of positively staining cells. The intensity of IR was stratified into four categories: 0, no IR; 1, weak IR; 2, moderate IR; and 3, strong IR. The proportion of positive cells was classified into four groups: 0, 0–5% tumor cells exhibiting IR; 0.33, 5–33% of the tumor cells exhibiting IR; 0.67, 33–67% of the tumor cells exhibiting IR; and 1, 67–100% of the tumor cells exhibiting IR.

Cell culture
Cell plates were precovered with Matrigel (BD Biosciences, Franklin Lakes, NJ, USA) at 5 µg/cm2. Human gastric cancer cell lines SGC7901 and MKN45, described previously (26) , were maintained on cell plates at 37°C, 5% CO2 in Dulbecco’s modified Eagle medium (DMEM) (GIBCO) supplemented with 10% FBS, 100 U/ml penicillin, and 0.1 mg/ml streptomycin.

Adhesion assay
The abilities of gastric cancer cells to adhere to Matrigel were determined in 24-well plates as described by others (27) . The plate surface was covered 0.2 ml of 50 µg/ml Matrigel, incubated for 2 h, and the supernatant was removed. A 0.5 ml suspension of tumor cells (1x105/ml) was transferred into the covered wells. After 0.5 h, 1 h, 2 h, and 4 h of incubation at 37°C, adhesive cells were washed with PBS twice, then counted under a microscope at x 200 magnification on 10 random fields in each well. Each experiment was performed in triplicate.

Invasion assay
Cell invasion assays were performed as described by others (28) using Transwells (8 mm pore size, Corning Costar Corp., Acton, MA, USA). Matrigel was diluted to a concentration of 2 mg/ml, and 50 µl of this solution was placed on the lower surface of a polycarbonate filter and air-dried. After being rinsed with PBS, the filters were placed into wells and 700 µl of DMEM containing 10% bovine serum was added into the lower compartment. Freshly trypsinized and washed cells (SGC7901 or MKN45) were suspended at 2 x 105/ml in DMEM containing 1% bovine serum and preincubated for 10 min with or without blocking antibodies. In some cases, inhibitors (PD98059, SP600125, or SB203580) were also added to the upper and lower chambers. The cell suspension (150 µl) was placed in upper compartments and the cells were allowed to invade for 24 h at 37°C in a 5% CO2 humidified incubator. After incubation, cells were removed from the upper surface of the filter with the cotton swab; the cells that had invaded the bottom surface of the filter were fixed with methanol and stained with hematoxylin. The invasiveness was determined by counting of the penetrating cells under a microscope at x200 magnification on 10 random fields in each well. Each experiment was performed in triplicate.

Tail vein metastatic assay
Mice were handled using best humane practices and were cared for in accordance with NIH Animal Care and Use Committee guidelines. Cells were harvested from tissue culture flasks using trypsin and washed three times with PBS. Mice were injected with 1 x 106 cells in 0.1 ml PBS through the tail vein. In some cases, mice were given 5 mg/kg PD98059 or MMP11 Ab suspended in 0.1 ml PBS through the tail vein each week after cell injection. The mice were then monitored for overall health, and total body weight. After 4 wk of injection, the mice were sacrificed. The liver tissues were observed with the naked eye and the number of visible tumors in liver surface was counted. Liver tissues were made into serial sections before being HE dyed and observed under a light microscope. Each experimental group contained 6 to 10 mice.

Plasmid construction and cell transfection
pSilencer3.0 (Ambion, Austin, TX, USA) was used for construction of human PrPc siRNA vectors PrPsi1 and PrPsi2 according to the manufacturer’s protocol. Two pairs of specific oligonucleotides (P1, P1', and P2, P2') were annealed, then subcloned into the BamHI/HindIII cloning site of pSilencer3.0, respectively. Full-length human PrP vector was constructed and described previously (6) . PrP{Delta}N, PrP{Delta}OR, and PrP{Delta}C were derived form full-length human PrPc by two-step overlapping polymerase chain reaction (PCR) with the general and overlapping primers. Taking PrP{Delta}N construction for an example, the general F1 primer and overlapping {Delta}N primer were used for generating the former fragment of PrP{Delta}N and the general R1 primer and overlapping {Delta}N' primer for the latter fragment. Then the two PCR fragments of PrP{Delta}N were purified and mixed for the second step PCR with general primers F1 and R1. The final PCR product was subcloned into pcDNA3.1B vector (Invitrogen, Carlsbad, CA, USA) with BamHI and EcoRI for producing a fusion protein with a C-terminal 6His tag. All primer sequences used for plasmid construction were listed in Table 1 . For construction of pGL3-MMP11, the promoter fragment (nucleotides –1468 to+28) was cut from 1.47ST3-chloroamphenicol acetyltransferase vector with KpnI and XhoI and subcloned into pGL3-enhancer vector (Promega, Madison, WI, USA). All the resulting plasmids were verified by direct DNA sequencing. Cell transfection was performed with Lipofectamine2000 (Invitrogen) as described in the manufacturer’s protocol. Briefly, cells were plated and grown to 70–90% confluence without antibiotics, then transfected with 1 µg plasmids. For transient transfection, cells were harvested for further experiments after 48 h of transfection. For stable transfecion, G418 (400 µg/ml) was added into cells after 24 h of transfection. Mixed clones were screened and expanded for an additional 6 wk. Gastric cancer cell line MKN45 transfected with PrPsi1, PrPsi2, and pSilencer was designated as MKN45-PrPsi1, MKN45-PrPsi2, and MKN45-cont, respectively. SGC7901 transfected with PrPsi1, PrPsi2, pSilencer, PrP{Delta}N, PrP{Delta}OR, PrP{Delta}C, and pcDNA3.1-V5/6His B was designated as SGC7901-PrPsi1, SGC7901-PrPsi2, SGC7901-cont, SGC7901-PrP{Delta}N, SGC7901-PrP{Delta}OR, SGC7901-PrP{Delta}C, and SGC7901-plasmid construct DNA, respectively.


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  		Table 1. Primers for plasmid construction

Western blot
Cells were washed twice with Hanks’s balanced salt solution and lysed directly in radio-immunoprecipitation assay buffer (50 mM Tris–HCl (pH 7.4), 1% (v/v) Triton X-100, 1 mM EDTA, 1 mM leupeptin, 1 mM phenylmethylsulfonyl fluoride, 10 mM NaF, 1 mM Na3VO4). The lysates were centrifuged at 14,000 rpm for 30 min at 4°C and the supernatants were collected. To detect the expression of the secreted active form of MMP11 in supernatant, 10 ml of conditioned medium was concentrated 100-fold in 10 kDa microcentrifuge concentrators (Millipore, Billerica, MA, USA). In some cases, cells were preincubated with 10 µM of PD98059 for 24 h. Cell lysate (60 µg) or supernatant proteins (10 µg) was separated by SDS-PAGE, blotted onto nitrocellulose membrane, and incubated with a primary Ab: anti-PrPc 3F4 (Sigma) diluted 1:2000, anti-6His (Abcam, Cambridge, MA, USA) diluted 1:1000, anti-MMP11 (Lab Vision, Fremont, CA, USA) diluted 1:500, antiphospho p38 MAPK (Cell Signaling Technology, Danvers, MA, USA) diluted 1:1000, antiphospho c-Jun NH2-terminal kinase (JNK) (Cell signaling Technology) diluted 1:2000, antiphospho ERK1/2 (Cell signaling Technology) diluted 1:3000, anti-ERK1/2 (Cell Signaling Technology) diluted 1:5000 or anti-ß-actin (Sigma) diluted 1:5000. After repeated washing, the membranes were incubated with horseradish peroxidase-conjugated anti-rabbit or antimouse secondary Ab (Santa Cruz Biotechnology, Santa, Cruz, CA, USA) diluted 1:2000. The bands were visualized using the enhanced chemiluminescence (ECL) system (Amersham Pharmacia Biotech, Arlington Heights, IL, USA).

RT-polymerase chain reaction (RT-PCR)
RT-PCR was performed as described previously (26) . Total cellular RNA was isolated from human cell lines using Trizol Reagent (Invitrogen). The concentration of RNA was determined through spectrophotometric measurement. cDNA was prepared using 2 µg of the RNA sample and a reverse transcription kit (MBI). MMP11 forward and reverse primers were 5'-CCC GCA ACC GAC AGA A-3' and 5'-GGG TAG CGA AAG GTG TAG AA-3', respectively, and gave an ~ 0.45 kb product. The housekeeping gene ß-actin was adopted as endogenous control for RNA normalization. ß-Actin forward and reverse primers were 5'-ATG ATA TCG CCG CGC TCG TC-3' and 5'-CGC TCG GTG AGG ATC TTC A-3', respectively, and gave an ~0.58 kb product. PCR was performed in a GeneAmp PCR system 2400 thermocycler (PERKin–Elmer, Norwalk, CT, USA). Conditions for PCR were 40 s at 94°C, 30 s at 55°C, and 40 s at 72°C (24 cycles). PCR products were loaded onto a 1.5% agarose gel and electrophoretically separated. The gel was then visualized under UV light after ethidium bromide staining.

Dual luciferase reporter assay
SGC7901 cells were plated at a density of 3 x 105/35 mm dish ~12 h before transfection. For transient transfections, 1.0 µg of total plasmid DNA was transfected into cells using Lipofectamine2000 Reagent (Invitrogen). 0.2 µg of the plasmid PGL3-MMP11 was always transfected and 0.02 µg of phRL-TK vector (Promega) was used as an internal control. Cotransfection experiments were performed with 0.2, 0.4, 0.8g of PrPsi1 plasmids or 0.6 µg of the deletion mutant vector PrP{Delta}N, PrP{Delta}OR, PrP{Delta}C or full-length vector PrP. PD98059 was incubated with concentration of 10 µM for 24 h before luciferase assays. After cultivation for 48 h, the transfected cells were harvested, lysed, centrifuged to pellet the debris, and subjected to the luciferase assay. Luciferase activity was measured as chemiluminescence in a luminometer (Perkin-Elmer, Norwalk, CT, USA) using the Dual-Luciferase Reporter Assay System (Promega) according to the manufacturer’s protocol. All transfections were performed in triplicate.

Statistical analysis
Each experiment was repeated at least three times. Bands from Western blot or RT-PCR were quantified by Quantity One software (Bio-Rad, Hercules, CA, USA). Relative protein or mRNA levels were calculated by referring them to the amount of ß-actin. Numerical data are presented as the mean ±SE. The difference between means was performed with ANOVA, then a post hoc test. All statistical analyses were performed using SPSS11.0 software (Chicago, IL, USA). P[lt]0.05 was considered as statistically significant.

RESULTS

PrPc is overexpressed in metastatic gastric cancer
In our previous study, we have shown that PrPc has a higher expression in gastric cancer tissues than adjacent nontumorous tissues and normal gastric mucosa (6) . To investigate the relationship between the expression of PrPc and metastasis of gastric cancer, we first compared the expression of PrPc in primary sites with corresponding metastatic sites of lymph node from 22 patients enduring metastatic gastric cancer. It was found that PrPc was predominantly located in the cytoplasm and membrane of gastric cancer cells (Fig. 1 A), which was consistent with our previous results. The average staining scores of primary site and corresponding metastatic site of lymph node were 2.23 ± 0.56 and 2.45 ± 0.64, respectively (Fig. 1B ). No significant difference in intensity of immunoreactivity or average staining score of PrPc was found between the primary and metastatic sites from the same patients (data not shown). We then compared expression of PrPc in primary sites from 86 patients enduring nonmetastatic gastric cancer with those from 38 patients enduring metastatic gastric cancer (Fig. 1A ). As shown in Table 2 , a positive rate of PrPc expression in nonmetastatic gastric cancer was 60.5% (52/86), lower than 92.1% (35/38) in metastatic gastric cancer. The average staining score in metastatic gastric cancer was significantly higher than that in nonmetastatic gastric cancer (2.69±0.73 vs. 1.22±0.47, P<0.05). These results suggested that PrPc was prone to highly expressed in metastatic gastric cancer but could not discriminate primary site from metastatic site from the same metastatic gastric cancer.


Figure 1
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  		Figure 1. Immunohistochemical analysis of PrPc in metastatic and nonmetastatic gastric cancers. A) Expression of PrPc was determined by immunohistochemical staining with PrPc Ab 3F4. a) noncancerous region of gastric cancer; b–c) primary site of nonmetastatic gastric cancer; d) primary site of metastatic gastric cancer; e) metastatic site of metastatic gastric cancer in lymph node; f) no primary Ab control. B) Results of immunohistochemical staining were evaluated by the staining scores described in Materials and Methods. *P < 0.05 vs. nonmetastatic gastric cancer.


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  		Table 2. Intensity of immunoreactivity of PrPc in metastatic and non-metastatic gastric cancer

PrPc promote adhesive, invasive, and in vivo metastatic abilities of gastric cancer cells
Our previous work has showed that PrPc was ubiquitously expressed in gastric cancer cell lines, including SGC7901 and MKN45 (6) . These two cell lines were demonstrated to have invasive ability to penetrate Matrigel-coated transwell in in vitro invasion assay and metastatic ability to metastasize mainly to liver instead of lung and other organs in in vivo tail vein metastatic assay (data not shown). To down-regulate the expression of PrPc in gastric cancer cells, two PrPc-specific siRNA vectors, named PrPsi1 and PrPsi2, were designed and constructed, aiming at 319–337 and 664–682 in the coding sequence of PrPc, respectively. After cell transfection and antibiotic screening for 6 wk, the expression of PrPc in stable transfected cells was determined by Western blot. PrPsi1 could down-regulate the expression of PrPc in SGC7901 and MKN45 effectively while the effect of PrPsi2 on PrPc expression was minimal (Fig. 2 A); the stable transfected SGC7901-PrPsi1 and MKN45-PrPsi1 were then chosen for further cellular assay.


Figure 2
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  		Figure 2. Effect of PrPc siRNA on adhesive, invasive, and metastatic abilities of gastric cancer cells. A) After stable transfection, the expression of PrPc was evaluated by Western blot. ß-Actin was used as an internal control. B) After 0.5 h, 1 h, 2 h, and 4 h of incubation, the cells attached to Matrigel were counted under a microscope. **P < 0.01 and *P < 0.05 vs. SGC7901-cont (upper panel) or MKN45-cont (lower panel). C) Invasive ability was evaluated by counting cells invading through matrigel and membrane with 8 µm pore of transwell. **P < 0.05 vs. cells transfected with pSilencer or mock. D) Mice were injected with 1 x 106 cells through tail vein. 4 wk later the mice were sacrificed. Liver tissues were observed with the naked eye and the number of visible tumors in liver surface was counted (left panel). The liver tissues were made serial sections before being HE dyed and observed under a light microscope (right panel). *P < 0.05 vs. cells transfected with pSilencer3.0.

To evaluate the effect of PrPc on cell adhesion, the ability of SGC7901-PrPsi1 and MKN45-PrPsi1 to adhere to matrigel (a solubulized basement membrane rich in ECM) was investigated by adhesive assay. All the gastric cancer cells bound to matrigel with a time-dependent manner. However, down-regulation of PrPc in SGC7901 and MKN45 decrease their adhesive ability to matrigel (Fig. 2B ). Since invasive potential is a common feature in the process of tumor metastasis, we next studied the influence of PrPc on the invasive ability of gastric cancer cells in vitro invasion assay. As shown in Fig. 2C , PrPc siRNA transfection produced a marked inhibition of invasion of SGC7901 and MKN45 through matrigel on Boyden chamber assay, with an average inhibiting rate of 56.4% and 43.5%, respectively. Tail vein metastatic assay in nude mice was further adopted to examine the in vivo metastatic ability of SGC7901-PrPsi1 and MKN45-PrPsi1. Compared with control cells transfected with empty vector, i.v. inoculation of SGC7901-PrPsi1 and MKN45-PrPsi1 cells led to significantly less visible tumors in liver surface (Fig. 2D , both P<0.05). Both in vitro invasion assay and in vivo nude mice assay suggested that PrPc had a potential to promote metastasis of gastric cancer.

MMP11 is involved in invasion of gastric cancer regulated by PrPc
ECM degradation is an essential step in tumor invasion and metastasis, which was mainly mediated by some matrix metalloproteinases, such as MMP2 and MMP9 (29) . Both previous study of microarray conducted between PRNP–/– and PRNP+/+ fibroblast cells by Satoh et al. (30) and our microarray data (data not shown) showed that MMP11 could be up-regulated by PrPc. Therefore, we examined the expression of MMP11 in gastric cancer cells after PrPc siRNA transfection. Our data confirmed that the expression of MMP11 in cytoplasm (inactive form, 55 kDa) and supernatant (active form, 45 kDa) of SGC7901 could both be down-regulated by PrPc siRNA (Fig. 3 A). However, PrPc did not alter the expression levels of MMP2 and MMP9 proteins. Furthermore, RT-PCR results showed that the expression level of MMP11 mRNA was also decreased in SGC7901-PrPsi1 compared to mock or empty vector transfected cells (Fig. 3A (right panel)). To investigate the possible mechanisms involved in regulation of MMP11 by PrPc, we performed dual luciferase reporter assay. The results showed that transfection of different contents of PrPc siRNA led to a 1.5- to 2.5-fold decrease of MMP11 promoter activity compared to empty vector transfection (Fig. 3B ), indicating that PrPc caused transactivation of MMP11 promoter and the consequent up-regulation of MMP11 mRNA and protein. To study the possible role of MMP11 in PrPc-related invasion, SGC7901-PrPsi1 and SGC7901-control cells were treated with MMP11 Ab (0.1 µg/ml or 1 µg/ml) before performing invasion assay. The results demonstrated that treatment with MMP11 Ab could inhibit the invasive activities of both cell lines. Inhibiting rates caused by MMP11 Ab at the concentration of 1 µg/ml in SGC7901 and SGC7901-cont were 50.3% and 57.4%, respectively, both were significantly higher than 30% in SGC7901-PrPsi1 cells (Fig. 3C ). The effect of MMP11 Ab was specific since the control IgG had no effect on invasion of gastric cancer cells. Taken together, we suggested that promoting the effect of PrPc on metastasis of gastric cancer was at least partially mediated by overexpression of MMP11 and possibly the consequent degradation of ECM.


Figure 3
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  		Figure 3. The inducible effect of PrPc on MMP11. A) The expression of MMP2, MMP9, MMP11 in cytoplasm and active form of MMP11 in supernatant was evaluated by Western blot (left panel). The expression of MMP11 mRNA was examined by RT-PCR (right panel). ß-Actin was used as an internal control. B) Relative luciferase activity of MMP11 promoter in SGC7901 cells cotransfected with PrPsi1 or empty vector was evaluated by dual luciferase reporter assay. **P < 0.01 vs. empty vector pSilencer. C) 2 x 105 cells were preincubated with IgG (1 µg/ml) or MMP11 Ab (0.1 µg/ml or 1 µg/ml), then subjected to invasion assay. **P < 0.01 and *P < 0.05 vs. mock and IgG treatment.

ERK1/2 signal is involved in gastric cancer invasion and MMP11 transactivation regulated by PrPc
It has been demonstrated that extracellular regulated kinase (ERK) was an important downstream effector of PrPc (31) . Thus, we examined whether the MAPKs pathway might be involved in PrPc-related invasion of gastric cancer. First, the phosphorylated status of three members of mitogen-activated protein kinases—p38 MAPKs, JNKs, and ERKs—in SGC7901, SGC7901-cont and SGC7901-PrPsi1 cells cultured on Matrigel at 5 µg/cm2 were evaluated by Western blot. The results showed that both phosphorylated ERK 1 and 2 were markedly down-regulated by PrPc siRNA transfection, while the expression levels of phosphorylated p38 MAPKs, phosphorylated JNKs, and total ERK protein were not altered (Fig. 4 A). We then investigated the influence of PD98059, an MEK specific inhibitor, on the expression of MMP11 and the invasive ability of gastric cancer. After treatment with PD98059 at a concentration of 10 µM, both the protein expression and transcription level of MMP11 were significantly down-regulated in gastric cancer cells (Fig. 4B, C ). These alterations were more significant in control cells than in SGC7901-PrPsi1 cells, indicating that PrPc increased promoter activity and the expression of MMP11 by activating phosphorylated ErK1/2. PD98059 could also inhibit the invasive abilities of SGC7901 cells with or without PrPc siRNA transfection. The inhibiting rates of PD98059 on invasive abilities of SGC7901 and SGC7901-cont cells were 44.2% and 62.2%, respectively, higher than 34.9% of SGC7901-PrPsi1 cells (Fig. 4D ). The expression of MMP11 and the invasive ability of SGC7901 were not significantly influenced by a p38 MAPK inhibitor SB203580 and a JNK inhibitor SP600125 (data not shown). All these results suggested that ERK1/2 kinases of MAPK family were specifically involved in PrPc-related invasion and mediated transactivation of MMP11 induced by PrPc.


Figure 4
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  		Figure 4. ERK signal in regulating invasiveness of gastric cancer cells induced by PrPc. A) The expression of phospho-p38 MAPK, phospho-c-Jun NH2-terminal kinase, phospho-ERK, total ERK was determined in gastric cacner cells by Western blot. ß-Actin was used as an internal control. B) After cells were treated with or without PD98059, the expression of secreted active form of MMP11 was examined by Western blot. ß-Actin was used as an internal control. C) After cells were treated with or without PD98059, the relative luciferase activity of MMP11 promoter in cells cotransfected with PrPsi1 or empty vector was evaluated by dual luciferase reporter assay. *P < 0.05 and **P < 0.01 vs. cells not treated PD98059. D) Invasion assay was conducted with or without adding PD98059 into upper or lower chambers of transwell. *P < 0.05 and **P < 0.01 vs. cells not treated with PD98059.

NH2-terminal of PrPc mediates the regulatory effect of PrPc on invasion of gastric cancer
Several regions of PrPc have been revealed to be key components in performing its function, especially regarding the copper binding ability of octarepeat domain (8) . To investigate which region of PrPc might contribute to the promoting effect of PrPc upon invasion of gastric cancer, three deletion mutant vectors of PrPc were constructed, including PrP{Delta}N, PrP{Delta}OR, and PrP{Delta}C, with deletions of N-terminal flexible domain (4–90), octarepeat domain (51–90), and C-terminal structured domain (96–230) in PrPc, respectively (Fig. 5 A). We further transfected SGC7901 with these deletion mutant vectors and the stable cell lines we obtained were designated SGC7901-PrP{Delta}N, SGC7901-PrP{Delta}OR, SGC7901-PrP{Delta}C, and SGC7901-PrP (transfected with full length of PrPc), respectively. We checked the expression of deletion mutants of PrPc in these stable cell lines. To exclude the interference of internal PrPc in SGC7901 when performing Western blot, the expression of external PrPc deletion mutants was detected by anti-6His Ab. As shown in Fig. 5B , all the 6His tagged mutants of PrPc could be detected whereas no signal was found in control cell transfected with empty vector.


Figure 5
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  		Figure 5. The effects of different deletion mutants of PrPc on invasiveness of gastric cancer. A) Full length of PrPc and deletion mutants PrP{Delta}N (24–90), PrP{Delta}OR (51–90) and PrP{Delta}C (96–230) were constructed. B) The expression of PrPc in cells was evaluated by Western blot with anti-6His Ab. The expression levels of phospho-ERK, total ERK, and secreted active form of MMP11 were also examined by Western blot. ß-Actin was used as an internal control. C) Invasive ability was evaluated by counting cells invading matrigel and membrane with 8 µm pore of transwell. Cells were pretreated with or without PD98059 or MMP11 Ab. *P < 0.05 vs. PrP, PrP{Delta}OR, and PrP{Delta}C; **P < 0.01 vs. pcDNA. D) Relative luciferase activity of MMP11 promoter in SGC7901 cells cotransfected with PrP, PrP{Delta}N, PrP{Delta}OR, PrP{Delta}C or empty vector was evaluated by dual luciferase reporter assay. Cells were pretreated with or without PD98059. *P < 0.05 vs. PrP, PrP{Delta}OR and PrP{Delta}C; **P < 0.01 vs. pcDNA. E) Mice were injected with 1 x 106 cells transfected with pcDNA, PrP, PrP{Delta}N, PrP{Delta}OR, or PrP{Delta}C through the tail vein, respectively. PD98059 or MMP11 Ab were injected through the tail vein each week after cell injection. Four weeks later, the mice were sacrificed and the number of visible tumors in liver surface was counted. *P < 0.05 vs. PrP, PrP{Delta}OR and PrP{Delta}C; **P < 0.01 vs. pcDNA.

When performing in vitro an invasion assay, we found that SGC7901-PrP, SGC7901-PrP{Delta}OR, and SGC7901-PrP{Delta}C showed a higher ability to invade than SGC7901-plasmid construct DNA. However, the invasive ability of SGC7901-PrP{Delta}N was significantly decreased compared to control cells (Fig. 5C ). This result indicated that N-terminal flexible domain was an important region conferring to PrPc-related invasion of gastric cancer. We further investigated the effect of N-terminal region on ERK signal and transcription of MMP11. The results showed that phosphorylated ERK1/2 and the active form of MMP11 in cells culture on Matrigel could be up-regulated by PrP, PrP{Delta}OR, or PrP{Delta}C transfection. However, they were down-regulated by PrP{Delta}N transfection (Fig. 5B ). The invasive abilities of all cell lines were declined after PD98059 or MMP11 Ab treatment, and the alteration was more significant in PrPc-overexpressed cell line SGC7901-PrP and SGC7901-plasmid construct DNA compared to SGC7901-PrP{Delta}N (34.0%, 29.2%, and 5%, respectively, after treated with PD98059; 55.3%, 40%, and 19.0% separately after being treated with MMP11 Ab). Our data also showed that the transcriptional activity of MMP11 promoter was markedly up-regulated by transfecting with PrP, PrP{Delta}OR, and PrP{Delta}C compared to empty vector. The effects of PrP, PrP{Delta}OR, and PrP{Delta}C on MMP11 promoter were abolished by PD98059 pretreatment. PrP{Delta}N transfection inhibited the transcriptional activity of MMP11 promoter (Fig. 5D ), the inhibiting rate of which was similar to PrPsi1 transfection (data not shown). The result of in vivo metastatic assay was similar to that of the invasion assay. Compared to empty vector plasmid construct DNA (pcDNA), PrP, PrP{Delta}OR, and PrP{Delta}C increased while PrP{Delta}N decreased the ability of SGC7901 to metastasize to mice liver (Fig. 5E ). The effects of PrP, PrP{Delta}OR and PrP{Delta}C on in vivo metastatic ability of SGC7901 were significantly antagonized by tail vein injection of PD98059 and MMP11 Ab. Taken together, these results indicated that N-terminal flexible domain was an important region of PrPc in activating ERK, regulating the transcriptional activity of MMP11, and consequently promoting the invasive and metastatic ability of gastric cancer.

DISCUSSION

In the present study, we presented the first evidence that PrPc had a high expression in metastatic gastric cancer and could promote the invasive activities of gastric cancer cells. This effect of N-terminal region of PrPc was at least partially mediated by MEK/ERK pathway and consequent transactivation of MMP11. To our knowledge, it is the first report to reveal the function of PrPc in invasiveness of cancers.

Metastasis is the final stage of tumor, characterized by the seeding and growth of satellite lesions in other organs (29 , 32) . Although the exact mechanisms of metastasis are not well-defined, it has been suggested that metastasis is not dependent on new genetic abnormalities that occur after tumors have been established and that the propensity to metastasize is determined early in the neoplastic process rather than near its end (22 , 33) . In the present study, no significant difference in PrPc expression was found between the primary site and the metastatic site of lymph node in metastatic gastric cancer. However, it was found to be highly expressed in metastatic gastric cancers compared to nonmetastatic ones. These data suggested that the alteration of PrPc expression was one of the early determinant events of metastasis, and PrPc may be used as a prognostic factor for metastasis of gastric cancer.

Matrix metalloproteinases (MMPs) are a family of metal-dependent enzymes that include collagenases, gelatinases, stromelysins, and membrane-type metalloproteinases, etc. (29) . They are the central components to degrade ECM and basement membrane during tumor progression. The stromelysins-3, also MMP11 (45 kDa and 55 kDa, active and inactive form), is a singular MMP whose substrate remains unknown (34) . MMP11 has been shown to be ectopically expressed in invasive human breast carcinoma (34) , nonsmall cell lung cancer (35) , colorectal cancer (36) , aggressive meningioma (37) , as well as gastric cancer (38) . The overexpression of MMP11 was often correlated with more aggressive phenotype and apoptosis-resistant features of tumors (39) . The present work demonstrated that blockage of MMP11 activity by Ab could partially reverse the promoting effect of PrPc on invasive abilities in gastric cancer cells by in vitro transwell assay. The positive role of MMP11 in promoting invasion showed here was consistent with a previous report that MMP11 might act as a tumor enhancer in processes leading to local invasion during progression of mouse mammary tumors (40) . It has been shown that MMP11 could be induced by cytokines and ECMs, such as interleukin (IL) -1beta, IL-2, TGF-beta, fibronectin, and collagen V (41 42) . Whether these molecules are involved in up-regulation of MMP11 caused by PrPc is not yet known.

A previous study has proposed that ERK1/2 are targets of PrPc-mediated signal in neuronal and non-neuronal cells. Ab-mediated ligation of PrPc elicits the modification of phosphorylation level of ERK1/2 (31) . As downstream effectors of oncogene Ras, the MEK/ERKs pathway has been demonstrated to be an important mediator of tumor progression and invasion (43) . Here we also demonstrated that PrPc in gastric cancer cells cultured on Matrigel could increase the expression level of phosphorylated ERK1/2. However, no significant alteration of phospho-ERK1/2 was found in PrP transfectants when no Matrigel existed in cell culture system (data not shown), which suggested that Matrigel was necessary for activation of MEK/ERK pathway and promoting invasive and metastatic abilities in gastric cancer cells by PrPc. The major components of Matrigel are laminin, collagen IV, heparan sulfate proteoglycans, and entactin. It has been found that at least two components of Matrigel—laminin, and heparan sulfate proteoglycans—can interact with PrPc. Therefore, it was possible that laminin, heparan sulfate proteoglycans, or both initiated the PrPc signal in gastric cancer cells. The present work also showed that the invasion promoting effects of PrPc could be inhibited by pharmacological blockade of MEK by PD98059, indicating MEK/ERK pathway played an important role in transducing invasion-promoting signal of PrPc in gastric cancer cells. It was found that Fyn governed all of the PrPc-induced pathways that converge to MEK/ERK module in neurons (44) . It was also likely that Fyn mediated the activation of ERK1/2 by PrPc in gastric cancer cells.

N-terminal fragment of PrPc is a flexible region that is highly conserved among different species, indicating this region is a putatively functional domain of PrPc (1) . In fact, amino acid residues 23–51 are essential for endocytosis, and the region 23–90 is sufficient to facilitate raft localization of PrPc (45 46) . The octarepeat region 50–90 of PrPc is a copper binding site, indicating a role of PrPc in copper metabolism and modulating a redox signaling. It had been shown that expression of the N-terminal fragment truncated PrP in the mouse led to ataxia and specific cerebellar lesions (47 48) . All these studies proposed that the N-terminal region was an important functional domain of PrPc. The present study suggested that N-terminal fragment of PrPc was a necessary region for promoting invasion of gastric cancer cells as well as ERK1/2 activation and MMP11 transactivation. It is not known yet how N-terminal fragment of PrPc initiated invasion-promoting signal in gastric cancer cells. Through the N-terminal domain, PrPc may transduce signal by binding to some molecules such as laminin receptor, caveolin, or neural cell adhesion molecules, which were never reported to interact with PrPc. Alternatively, intracellular endocytosed PrP could interact with the adaptor protein Grb2 (49) , an upstream regulator of Ras/Raf/MEK/ERK pathway, and then may transduce its signal of promoting invasiveness.

In conclusion, we provide evidence that PrPc is highly expressed in metastatic gastric cancer tissues, and the data strongly indicate that the N-terminal region of PrPc exhibits an invasion-promoting effect on gastric cancer cells at least in part by a mechanism involving the activation of MEK/ERK pathway and transactivation of MMP11. To our knowledge, the present work links for the first time PrPc to invasive abilities of cancer cells.

ACKNOWLEDGMENTS

We are grateful to Patrick Anglard for 1.47ST3-CAT vector and Chen Yu for the MMP2 and MMP9 monoclonal antibodies. We are also grateful to Zhang Jian for help in luciferase activity analyses, Qiao Taidong for excellent guide in the process of experiment.

FOOTNOTES

1 These two authors contributed equally to this work.

Received for publication March 14, 2006. Accepted for publication April 17, 2006.

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