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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
SPECIFIC AIM
The function of cellular prion protein (PrPc) is not well-defined. Here we investigated the possible role of PrPc in the process of metastasis in gastric cancer.
PRINCIPAL FINDINGS
1. PrPc is overexpressed in metastatic gastric cancer
To investigate the relationship between the expression of PrPc and metastasis of gastric cancer, we compared expression of PrPc in primary sites from 86 patients enduring nonmetastatic gastric cancer with those from 38 patients enduring metastatic gastric cancer. It was found that PrPc was predominantly located in the cytoplasm and membrane of gastric cancer cells. The 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 be highly expressed in metastatic gastric cancer.
2. PrPc promote adhesive, invasive, and in vivo metastatic ability of gastric cancer cells.
To down-regulate the expression of PrPc in gastric cancer cells, two PrPc-specific siRNA vectors, named PrPsi1 and PrPsi2, were designed and constructed. After cell transfection and antibiotic screening for 2 months, the expression of PrPc in stable transfected cells was determined by Western blot. PrPsi1 could down-regulate the expression of PrPc in gastric cancer cells SGC7901 and MKN45 effectively while the effect of PrPsi2 on PrPc expression was minimal. Down-regulation of PrPc in SGC7901 and MKN45 decrease their adhesive ability to matrigel. PrPc siRNA transfection also produced a marked inhibition of invasion of SGC7901 and MKN45 through matrigel upon 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. Both in vitro invasion assay and in vivo nude mice assay suggested that PrPc have a potential to promote metastasis of gastric cancer.
3. MMP11 is involved in invasion of gastric cancer regulated by PrPc
A previous study of microarray conducted between PRNP–/– and PRNP+/+ fibroblast cells by Satoh et al. and our microarray data both showed that MMP11 could be up-regulated by PrPc. Here we 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. However, PrPc did not alter the expression levels of MMP2 and MMP9 proteins. The expression level of MMP11 mRNA was also decreased in SGC7901-PrPsi1 compared to mock or empty vector transfected cells. The dual luciferase reporter assay demonstrated 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, indicating that PrPc caused transactivation of MMP11 promoter, then the 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 antibody (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 significantly higher than 30% in SGC7901-PrPsi1 cells. 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 effect of PrPc on metastasis of gastric cancer be at least partially mediated by overexpression of MMP11 and possible consequently degradation of extracellular matrix (ECM).
4. ERK1/2 signal is involved in gastric cancer invasion and MMP11 transactivation regulated by PrPc
Both phosphorylated ERK 1 and 2 could be markedly down-regulated by PrPc siRNA transfection, while the expression levels of phosphorylated p38 MAPKs, phosphorylated JNKs, and total ERK protein were not altered. The influence of PD98059, an MEK specific inhibitor, on the expression of MMP11 and invasive ability of gastric cancer was then evaluated. After treatment with PD98059 at a concentration of 10 µM, both the protein expression and transcriptional level of MMP11 were significantly down-regulated in gastric cancer cells. These alterations were more significant in control cells than in SGC7901-PrPsi1 cells. PD98059 could also inhibit the invasive abilities of SGC7901 cells with or without PrPc siRNA transfection. The inhibiting rates of PD98059 on invasive ability of SGC7901 and SGC7901-cont cells were 44.2% and 62.2%, respectively, higher than 34.9% of SGC7901-PrPsi1 cells. All these results suggested that ERK1/2 kinases of MAPK family are specifically involved in PrPc-related invasion and mediate transactivation of MMP11 induced by PrPc.
5. NH2-terminal of PrPc mediates the regulatory effect of PrPc on invasion of gastric cancer
To investigate which region of PrPc might contribute to the promoting effect of PrPc on invasion of gastric cancer, three deletion mutant vectors of PrPc were constructed, including PrP
N, PrPOR, and PrPC, with deletion of an N-terminal flexible domain (24–90), octarepeat domain (51–90), and C-terminal structured domain (96–230) in PrPc, respectively. We then transfected SGC7901 with these vectors and performed an in vitro invasion assay, and found that SGC7901-PrP, SGC7901-PrPOR, and SGC7901-PrPC had a higher ability to invade than SGC7901-cont. However, the invasive ability of SGC7901-PrPN was significantly decreased compared to control cells. 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 the N-terminal region on ERK signal and transcription of MMP11. The results showed that phosphorylated ERK1/2 and an active form of MMP11 in cells culture on Matrigel could be up-regulated by PrP, PrPOR, or PrPC transfection. However, they were down-regulated by PrPN transfection. The invasive abilities of all cell lines were declined after PD98059 or MMP11 Ab treatment, and the alteration was more significant in PrPc-overexpressing cell line SGC7901-PrP, then SGC7901-cont, compared to SGC7901-PrPN (34.0%, 29.2%, and 5%, respectively, after treatment with PD98059; 55.3%, 40%, and 19.0% separately after treatment with MMP11 Ab). Our data also showed that the transcriptional activity of MMP11 promoter was markedly up-regulated by transfecting with PrP, PrPOR, and PrPC compared to empty vector. The effects of PrP, PrPOR, and PrPC on MMP11 promoter were abolished by PD98059 pretreatment. PrPN transfection inhibited the transcriptional activity of MMP11 promoter. The result of in vivo metastatic assay was similar to that of invasion assay. Compared to empty vector plasmid construct DNA (pcDNA), PrP, PrPOR, and PrPC increased while PrPN decreased the ability of SGC7901 metastasizing to mice liver. The effects of PrP, PrPOR, and PrPC 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.
CONCLUSIONS AND SIGNIFICANCE
In the present study, no significant difference of PrPc expression was found between primary site and 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 is an early determinant of metastasis, and PrPc may be used as a prognostic factor for metastasis of gastric cancer.
The overexpression of MMP11 was often correlated with more aggressive phenotype and apoptosis-resistant feature of tumors. The present work demonstrated that blockage of MMP11 activity by Ab could partially reverse the promoting effect of PrPc on invasive ability in gastric cancer cells. 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. 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. Whether these molecules are involved in up-regulation of MMP11 caused by PrPc is not known yet.
An earlier study proposed that ERK1/2 are targets of PrPc-mediated signal in neuronal and nonneuronal cells. 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, which suggested that Matrigel was necessary for activation of the MEK/ERK pathway by PrPc. It has been found that at least two components of Matrigel—laminin and heparan sulfate proteoglycans—can interact with PrPc. Therefore, it is possible that laminin, heparan sulfate proteoglycans, or both initiated the PrPc signal in gastric cancer cells. Fyn was thought to govern all the PrPc-induced pathways that converge to MEK/ERK module in neurons. It is likely that Fyn mediates the activation of ERK1/2 by PrPc in gastric cancer cells.
N-terminal region an important functional domain of PrPc. The present study demonstrated that the N-terminal fragment of PrPc was an indispensable region for promoting invasion of gastric cancer cells as well as ERK1/2 activation and MMP11 transactivation. It is not yet known how the N-terminal fragment of PrPc initiated invasion-promoting signal in gastric cancer cells. Through the N-terminal domain, PrPc may transduce the signal by binding to some molecules, such as laminin receptor, caveolin, or neural cell adhesion molecules, which have been reported to interact with PrPc before. Alternatively, intracellular endocytosed PrP can interact with the adaptor protein Grb2, an upstream regulator of Ras/Raf/MEK/ERK pathway, 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 activation of the MEK/ERK pathway and transactivation of MMP11 (Fig. 1
). To our knowledge, the present work, for the first time, links PrPc to invasive ability of cancer cells.
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FOOTNOTES
1 These authors contributed equally to this work. 
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.06-6138fje
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