Silencing of C-Met by RNA Interference Inhibits the Survival, Proliferation, and Invasion of Nasopharyngeal Carcinoma Cells

Silencing of c-Met by RNA interference inhibits the survival, proliferation, and invasion of nasopharyngeal carcinoma cells

Yuncheng Li, Sulin Zhang, Zhengang Tang, Jian Chen & Weijia Kong

Tumor Biology Tumor Markers, Tumor Targeting and Translational Cancer Research

ISSN 1010-4283 Volume 32 Number 6

Tumor Biol. (2011) 32:1217-1224 DOI 10.1007/s13277-011-0225-y

1 23 Your article is protected by copyright and all rights are held exclusively by International Society of Oncology and BioMarkers (ISOBM). This e-offprint is for personal use only and shall not be self-archived in electronic repositories. If you wish to self-archive your work, please use the accepted author’s version for posting to your own website or your institution’s repository. You may further deposit the accepted author’s version on a funder’s repository at a funder’s request, provided it is not made publicly available until 12 months after publication.

1 23 Author's personal copy

Tumor Biol. (2011) 32:1217–1224 DOI 10.1007/s13277-011-0225-y

RESEARCH ARTICLE

Silencing of c-Met by RNA interference inhibits the survival, proliferation, and invasion of nasopharyngeal carcinoma cells

Yuncheng Li & Sulin Zhang & Zhengang Tang & Jian Chen & Weijia Kong

Received: 2 July 2011 /Accepted: 10 August 2011 /Published online: 16 September 2011 # International Society of Oncology and BioMarkers (ISOBM) 2011

Abstract c-Met is a tyrosine kinase receptor that mediates distributions [1]. The morbidity of NPC is 30–80/100,000 pleiotropic cellular responses following its activation by per year in Southern China and Southeast Asia, approxi- hepatocyte growth factor. The overexpression of c-Met in mately 30 times higher than elsewhere [2]. Unlike other nasopharyngeal carcinoma (NPC) has been described head and neck tumors, NPC is characterized by aggressive recently, but the functional role of c-Met in NPC remains invasion and distant metastasis [3]. Moreover, more than incompletely understood. This study aimed to investigate half of NPC cases are diagnosed at an advanced stage, the potential mechanism by which c-Met contributes to the which results in a high rate of treatment failure [4]. It has tumorigenesis of NPC. In the present study, by using RNA been reported that radiotherapy and chemotherapy for the interference we silenced the expression of c-Met in CNE-2 management of advanced NPC is useful [5, 6]. However, cells, a poorly differentiated NPC cell line. Our in vitro the prognosis of NPC is poor, with 5-year survival rate is studies showed that shRNA-mediated depletion of c-Met only about 50–60%, due to the high frequency of distant resulted in the suppression of proliferation, migration, and metastasis and local recurrence [1]. invasion, as well as an increase in the apoptosis of CNE-2 NPC is a malignant tumor that is well known to be cells. Moreover, in xenograft nude mice we demonstrated associated with Epstein–Barr virus infection [7]. A genetic that the depletion of c-Met resulted in reduced tumor predisposition for NPC has also been suggested, considering growth and increased apoptosis in xenografts. Taken the high incidence of NPC in patients with specific histocom- together, these results suggest that c-Met plays an onco- patibility complex profiles, including HLA-A2, HLA-B Sin2 genic role in the development of NPC and reveal it as a alleles, or HLA haplotypes Aw19, Bw46, and B17 [8]. potential novel therapeutic target for NPC. Additionally, RASSF1, a tumor suppressor that mediates the apoptotic effects of oncogene Ras, is frequently hyper- Keywords c-Met . Nasopharyngeal carcinoma . methylated in NPC [9]. However, the molecular pathogen- Proliferation . Invasion . Apoptosis esis of NPC is still not completely understood. c-Met is a prototypic member of receptor tyrosine kinases family which acts as the only known high-affinity Introduction receptor for hepatocyte growth factor (HGF) [10]. HGF is a multifunctional growth factor that plays an important role in Nasopharyngeal carcinoma (NPC) is a distinctive type of angiogenesis and tumor growth [11]. Upon HGF stimulation, head and neck cancer with particular racial and geographic c-Met induces several biological responses that collectively give rise to invasive growth of cancer [12]. Aberrant c-Met Yuncheng Li and Sulin Zhang contributed equally to this work. overexpression triggers tumor growth and the formation of Y. Li : S. Zhang : Z. Tang : J. Chen : W. Kong (*) new blood vessels that support tumor survival and metastasis Department of Otolaryngology, [13]. Importantly, high expression of c-Met was observed in Union Hospital of Tongji Medical College, 82.9% (112/135) of NPC tissues, and there was a positive Huazhong University of Science and Technology, correlation between c-Met expression and lymph node 1277 Jiefang Avenue, Wuhan, China 430022 metastasis [14]. Increased c-Met and/or HGF expression is e-mail: [email protected] often associated with low differentiation and poor prognosis Author's personal copy

1218 Tumor Biol. (2011) 32:1217–1224 of the tumor [15]. Very recent study showed that c-Met Dulbecco’s modified Eagle’s medium (DMEM, Gibco, tyrosine kinase inhibitors downregulated c-Met phosphory- USA) without phenol red, supplemented with 10% fetal lation and led to marked inhibition of the growth and bovine serum (FBS, Gibco) and nonessential amino acids, invasion of NPC cells [16]. Zhou also identified the in a 37°C incubator with humidified air containing 5% downregulation of Met by small interfering RNA decreased CO2. CNE-2 cells were grown to a confluence of 50–70%, tumor cell invasion/migration in vitro [17]. However, no and transfection was performed using Lipofectamine 2000 study has addressed the effects of loss of c-Met via RNA (Invitrogen, Carlsbad, CA) according to the manufacturer’s interference on the proliferation and apoptosis in CNE-2, a instructions. The cells stably transfected with recombinant poorly differentiated NPC cell line. In addition, no compre- c-Met shRNA plasmids were selected and placed into hensive research both in vitro and in vivo concerning c-Met DMEM containing G418 at a final concentration of 300 μg/mL biological function in NPC lines is available by now. for 8 weeks. The stable transfected cells (ShC1, ShC2, and Therefore, this study aimed to investigate the potential ShC3) were screened by limiting dilution assay. The stable mechanism by which c-Met contributes to the tumorigenesis transfected single cell clones, in which the fluorescence could of NPC. We employed NPC CNE-2 cells as the experimental last for 15 generations, were tested by Western blot for the model and found that reducing c-Met expression via RNA c-Met level. Cells transfected with a scrambled shRNA (Mock) interference in CNE-2 cells led to the reduced proliferation, and untreated CNE-2 cells (Blank) were regarded as the control invasion, and increased apoptosis of NPC cells. More groups. importantly, we validated the above in vitro results in xenograft nude mice and demonstrated that knockdown of Western blot analysis c-Met suppressed the tumorigenicity of CNE-2 cells in vivo. Cells in the logarithmic growth phase, were washed twice with cold phosphate-buffered saline (PBS), and lysed by Materials and methods sonication in 2% SDS buffer. The protein level of the lysate was measured with a BCA protein assay kit (Beyotime, c-Met short-hairpin RNA sequences and plasmid construction China) according to the manufacturer’s instructions. Fifty micrograms of lysate per lane was separated on 8% sodium Three candidate shRNA sequences directed against human dodecyl sulfate-polyacrylamide gel electrophoresis and c-Met mRNA (GenBank accession no. NM_001127500) transferred to nitrocellulose membranes (Millipore, USA) were designed. The details of these sequences were shown using a Biorad Mini-Transblot Apparatus (Biorad, Hercules, in Table 1. These 21-nucleotide (nt) sequences showed no CA). After blocking for 1 h at room temperature in a blocking homology with other known genes in the human genome buffer containing 0.1 M Tris–HCl, 0.9% NaCl, 0.5% Tween- by Blast analysis. Recombinant plasmids (pGPU6/GFP/ 20 (TBST), and 5% nonfat dry milk, the membranes were Neo-c-Met shRNA) were constructed by Shanghai incubated with primary rabbit monoclonal antibody against c- MingHong Biotech (Shanghai, China). Met (Abcam, Cambridge, UK, 1:2,000 dilution) in blocking buffer overnight at 4°C. Membranes were washed three times Cell culture and transfection for 10 min in TBST, and incubated with HRP conjugated goat anti-rabbit IgG antibody (Sigma-Aldrich, St Louis, MO, The human NPC cell line, CNE-2, was obtained from the 1:2,000 dilution) in blocking buffer for 1 h at room Institute of Biochemistry and Cell Biology, Chinese temperature. The membranes were developed by enhanced Academy of Sciences (Shanghai, China). Neither herpes- chemiluminescence (Amersham) on autoradiography film like virus particles nor Epstein–Barr virus capsid antigens (Kodak, Rochester, NY). The density of the bands was were found in the CNE-2 cell line. The cells were grown in quantified using the Image J software. The expression ratio

Table 1 The details of c-Met short-hairpin RNA (shRNA) used in this study shRNA Targeted c-Met mRNA sequence Loop Reverse complement sequence Termination Position in GenBank signal (NM_001127500) shC1 GGAAAGAACCTCTCAACAT TTCAAGAGA ATGTTGAGAGGTTCTTTCC TTTTTGGAT 2460 shC2 GCAACAGCTGAATCTGCAA TTCAAGAGA TTGCAGATTCAGCTGTTGC TTTTTGGAT 2659 shC3 GCAGTGAATTAGTTCGCTA TTCAAGAGA TAGCGAACTAATTCACTGC TTTTTGGAT 3135 Mock TTCTCCGAACGTGTCACGT TTCAAGAGA ACGTGACACGTTCGGAGAA TTTTTGGAT

The hairpin structure is composed of 21 pairs of complementary bases, a loop including nine oligonucleotides, and a termination sequence Author's personal copy

Tumor Biol. (2011) 32:1217–1224 1219

(c-Met/GAPDH) was used to evaluate c-Met protein expres- Mock, and ShC2) were harvested and single cell suspension. Each experiment was carried out in triplicate. sions (3×106 cells in 0.2 ml of Hanks solution) were injected subcutaneously into the right flank of the nude In vitro cell growth assay mice. The tumor diameter was measured and the tumor volume was calculated twice weekly by the formulas: ab2/2, Cells in the log-growth phase were harvested and sus- a and b as the two maximum diameters measured by a pended cells were seeded into 96-well plates at a density of sliding caliper. Four weeks later, the mice were killed and the 1×104 cells per well in 100 μL DMEM supplemented with expressions of c-Met in the tumor tissues were detected by 10% FBS. The medium was changed daily. After culture Western blot analysis. for 24 h, the number of viable cells was determined daily using the Cell Counting Kit-8 (CCK-8, Dojindo, Japan). TUNEL staining Briefly, 10 μL of the CCK-8 solution was added to each well, and after 4 h incubation at 37°C, the absorbance at Xenografts were excised from the nude mice, the formalin- 450 nm was measured by a microplate reader (μQuant, Bio- fixed and paraffin-embedded samples were cut in 4 μm Tek, USA). Each experiment was repeated in triplicate. sections at 1–2 mm intervals. TUNEL staining was performed with TUNEL Apoptosis Detection Kit (Milli- In vitro invasion and migration assay pore, USA) according to the manufacturer’s protocols. The 4-μm sections in slides were deparaffinized with xylene Each transwell chamber was coated with 30 μL matrigel and rehydrated with a graded series of ethanol. Endogenous (50 mg/L, 1:2 dilution, BD, USA), and polymerized at peroxidase activity was blocked by incubation in 3% 37°C for 30 min. Cells were collected into DMEM hydrogen peroxide for 30 min. Then, the sections were supplemented with 1% FBS. For each transwell chamber, treated with 0.01 mM sodium citrate buffer (pH 6.0) for 100 μL cell resuspension containing 2.0×105 cells was 15 min in a microwave oven at 95–100°C for the antigen added in the upper chamber. The lower chamber contained retrieval. After washing twice with PBS, the sections were 500 μL DMEM supplemented with 20% FBS. After 24 h at incubated with terminal deoxyribonucleotidyl transferase 37°C, cells and Matrigel in the upper well were removed. enzyme at 37°C for 1 h. The sections were washed three The cells in the lower chamber were stained with 1% times in PBS for 1 min at room temperature. After washing crystal violet, and then counted under the light microscope. twice with PBS, immunohistochemical reactions were In each well, the values of five randomly chosen visual visualized with diaminobenzedine (DAB kit, Boster, fields were averaged and defined as the cell numbers for the Wuhan, China). Then, the tissues were counterstained with well. All experiments were repeated in triplicate. The hematoxylin for 30 s, dehydrated with a graded series of migration assay was performed similar to the transwell ethanol and mounted. Apoptotic cells (approximately invasion assay, except that the transwell chamber was not 100 cells/field for three non-overlapping fields) were coated with Matrigel. counted. Apoptosis index was calculated as the percentage of apoptosis cells over total counted cells. Flow cytometry assay Statistical analysis Cells were collected and washed twice with PBS, and resuspended in PBS. Three hundred microliters of cell All data were expressed as the mean ± standard deviations. The suspension was mixed with 5 μL annexin V-FITC (Jingmei, statistical analysis was performed by employing SPSS 12.0 China) and 5 μL PI (20 μg/μL). The mixture was incubated software package (SPSS Inc, Chicago, IL). Student’s t test was at room temperature in the dark for 10 min. Cell apoptosis used to compare two groups and one-way analysis of was examined by using FACS Calibur (BD, USA). variance was used to compare multiple groups. p <0.05 was considered statistically significant. Tumorigenicity in nude mice

Thirty-six 4- to 6-week-old male athymic nude mice Results (BALB/c-nu) were obtained from the Experimental Animal Center of Huazhong University of Science and Technology shRNA-mediated depletion of c-Met in CNE-2 cells (Wuhan, China). All animals were housed under pathogen- free conditions and maintained according to the guidelines To confirm that c-Met shRNA vectors could deplete c-Met of the Committee on Animals Use of Huazhong University in NPC CNE-2 cells, we performed Western blot analysis to of Science and Technology. Three groups of cells (Blank, detect the protein level of c-Met in CNE-2 cells transfected Author's personal copy

1220 Tumor Biol. (2011) 32:1217–1224 with vectors harboring different targeting sequences against c-Met. The results showed that compared to the Mock, protein level of c-Met was significantly reduced after transfection of the c-Met shRNA recombinant plasmids (p=0.023). In addition, shC2 exerted the strongest effect in suppressing c-Met expression, with weaker effects exhibitedbyshC1andshC3(Fig.1). Therefore, we selected shC2 for the following experiments.

Depletion of c-Met attenuates the proliferation of CNE-2 cells

The proliferation of CNE-2 cells was assessed by CCK-8 assay and the results showed that the growth rate of shC2 stably transfected cells was significantly slower than that in the Mock cells (p=0.031 at 72 h and p=0.027 at 96 h, Fig. 2). These results indicate that c-Met promotes Fig. 2 Depletion of c-Met attenuates the proliferation of CNE-2 cells. The proliferation of CNE-2 cells was analyzed by CCK-8 assay. Blank: untreated CNE-2 cells; Mock CNE-2 cells transfected with a scrambled shRNA; shC2 CNE-2 cells transfected with c-Met shRNA2. The data were presented as average±standard error. Each experiment was done in triplicate. *P<0.05, vs. Mock at 72 and 96 h, respectively

the proliferation in CNE-2 cells and its suppression could lead to the inhibition of cell growth.

Depletion of c-Met inhibits the migration and invasion of CNE-2 cells

Next we performed in vitro migration and invasion assay and the results showed that the number of cells that passed through the membrane was 119.53±9.22, 94.30±6.83, and 56.70±9.31 in the Blank, Mock, and shC2 group, respectively. The number of cells migrated was significantly lower in shC2 group than in the Mock group (p=0.001, Fig. 3a). Moreover, the number of cells that passed through the transwell chamber polycarbonate membrane was 117.50±14.06, 90.25± 9.43, and 51.50±10.75 in the Blank, Mock, and shC2 group, respectively. Compared to Mock group, the number of cells passed the matrigel was significantly lower in shC2 group (p=0.000, Fig. 3b). Collectively, these results suggest that c-Met promotes the migration and invasion of CNE-2 cells. Fig. 1 shRNA-mediated knockdown of c-Met in CNE-2 cells. Untreated CNE-2 cells and the derived stable cells as indicated were Depletion of c-Met promotes the apoptosis of CNE-2 cells collected and subjected to Western blot analysis. a Representative blots showing the expression level of c-Met in CNE-2 cells of five Apoptosis ratio of CNE-2 cells was evaluated by flow groups. GAPDH served as loading control. b Photodensitometric analysis of relative expression level of c-Met after nominalization to cytometry assay. The apoptosis rate was 6.6±1.23%, 8.0± GAPDH. The data were presented as average±standard error. Each 1.17%, and 22.7±2.06% in the Blank, Mock, and shC2 experiment was done in triplicate.*P<0.05, vs. Mock. Blank: group, respectively, with significant difference between Mock untreated CNE-2 cells; : CNE-2 cells transfected with a shC2 group and Mock group (p=0.034, Fig. 4). These scrambled shRNA; shC1: CNE-2 cells transfected with c-Met shRNA1; shC2: CNE-2 cells transfected with c-Met shRNA2; shC3: results indicate that c-Met antagonizes the apoptosis of CNE-2 cells transfected with c-Met shRNA3 CNE-2 cells. Author's personal copy

Tumor Biol. (2011) 32:1217–1224 1221

Fig. 3 Depletion of c-Met inhibits the migration and invasion of CNE-2 cells. a Photo- graphs of migrated and invaded cells from three groups (×200 magnification). b Comparison of the cell migration and invasion activities among three groups. Blank: untreated CNE-2 cells; Mock: CNE-2 cells transfected with a scrambled shRNA; shC2: CNE-2 cells transfected with c-Met shRNA2. The data were presented as average±standard error. Each experiment was done in triplicate. *P<0.01, vs. Mock

Depletion of c-Met inhibits the in vivo tumorigenicity In addition, we performed TUNEL staining on the tumor of CNE-2 cells xenografts and found that the apoptotic index was 11.63± 3.41%, 13.15±4.07%, and 27.23±6.15% in the Blank, To validate the above in vitro results in an in vivo setting, Mock, and shC2 group, respectively, with significant we established xenograft NPC tumors by subcutaneous difference between shC2 group and Mock group injection of shC2 stably transfected CNE-2 cells or the (p=0.037, Fig. 5d). Taken together, these in vivo data corresponding Blank or Mock control cells into the flanks support the in vitro findings and suggest that c-Met of BALB/c-mu nude mice. In Blank or Mock group, the enhances the tumorigenicity of CNE-2 cells by promoting cells grew rapidly and formed palpable tumors at 5–7 days their growth and antagonizing their apoptosis. after the injection. In contrast, the tumor formation was remarkably slower after the injection of ShC2 stably transfected CNE-2 cells and the diameters of the tumors Discussion were significantly smaller (Fig. 5a, b). Western blot analysis confirmed that c-Met expression at protein level was c-Met is a tyrosine kinase receptor for HGF and overex- significantly reduced in tumor xenografts derived from pressed in a variety of malignant tumors [18], such as stably transfected CNE-2 cells (p=0.027, Fig. 5c). chordomas [19], gastrointestinal tract [20], and thyroid Author's personal copy

1222 Tumor Biol. (2011) 32:1217–1224

abBlank Mock ShC2 30 * 25

Apoptosis Ratio (%) 5

0 Blank Mock ShC2

Fig. 4 Depletion of c-Met promotes the apoptosis of CNE-2 cells. a scrambled shRNA; shC2: CNE-2 cells transfected with c-Met Flow cytometry analysis of apoptosis ratio in three groups. b shRNA2. The data were presented as average±standard error. Each Comparison of the apoptosis ratio among three groups. Blank: experiment was done in triplicate. *P<0.05, vs. Mock untreated CNE-2 cells; Mock: CNE-2 cells transfected with a

[21]. Our in vitro study showed that shRNA-mediated knockdown of c-Met led to increased apoptosis of CNE-2 depletion of c-Met resulted in the suppression of prolifer- cells both in vitro and in vivo, and significantly reduced ation, migration, and invasion. growth of CNE-2 xenografts in vivo. In the present In this study, we employed NPC CNE-2, a poorly study, our results showed that the depletion of c-Met differentiated NPC cell line, to show that shRNA- promoted the apoptosis of CNE-2 cells in vitro. mediated knockdown of c-Met obviously decreased the Moreover, the apoptotic index was higher in tumor growth of CNE-2 cells in vitro, consistent with previous tissues derived from c-Met knockdown CNE-2 cells results obtained by using c-Met chemical inhibitors [16]. than in those from control cells. It has been demon- Moreover, Xie et al., revealed that reduction the expres- strated that c-Met is a key regulator of cell survival and sion of c-Met significantly inhibited proliferation of apoptosis [30], but bidirectional regulation of apoptosis human hepatocellular carcinoma cell line [22]. These by c-Met do exist in tumor cells. c-Met inhibitor AM7 was results indicate that c-Met plays a crucial role in able to downregulate p53 and induce the apoptosis in NPC promoting NPC. cell lines [17]. On the other hand, c-Met inhibitor NK4 The ability of cancer cells to migrate and invade the promoted the apoptosis of colon cancer LoVo cells via the surrounding tissues is a critical step for tumor metasta- p38 MAPK pathway [31]. While HGF could prevent the sis [23]. Many studies have shown that the migration of apoptosis of HL7702 cells induced by the actinomycin D, cancer cells is associated with their invasive ability [24, which was associated with the activation of PI3K/Akt 25]. Our data showed that the inhibition of c-Met signaling [32]. Therefore, it is important to identify expression by shRNA could inhibit the migration and whether c-Met also promotes the apoptosis of CNE-2 invasion of NPC cells. Our results were in agreement with cells via the activation of p38 MAPK pathway or PI3K/ the previous reports. Kermorgant et al. revealed that Akt pathway in our future investigation. Taken together, activation of HGF/c-Met signaling has been shown to our results provide new evidence that c-Met promotes the increase the motility of Madin-Darby canine kidney cells tumorigenesis of NPC by antagonizing the apoptosis of and the invasion of Caco-2 colon cancer epithelial cells nasopharyngeal cancer cells. [26, 27]. In addition, the inhibitors of HGF/c-Met pathway It is worth noting that even after shRNA-mediated have been suggested to be effective in preventing cancer depletion of c-Met, CNE-2 cells still retained the capabil- invasion and metastasis [28, 29]. Further mechanism ities of proliferation and invasion. This may reflect the research is needed to elucidate the signaling pathway by incomplete depletion of c-Met in CNE-2 cells. Neverthe- which c-Met promotes the invasion and metastasis of less, it is also possible that other factors, such as vascular NPC. endothelial growth factor, urokinase type plasminogen Our study showed that shRNA-mediated depletion of c- activator, and matrix metalloproteinases, contribute to the Met resulted in an increase in the apoptosis of CNE-2 cells. malignant phenotypes of CNE-2 cells. Further analysis of To our knowledge, we were the first to show that the crosstalk between c-Met and these factors will be Author's personal copy

Tumor Biol. (2011) 32:1217–1224 1223

ac 7000 Blank Mock ShC2 Blank 6000 Mock c-Met 5000 ShC2

4000 3000 GAPDH 2000

Tumor volume (mm3) 1000 0.8 0 0.7 14 16 18 20 22 24 26 28 33 b Days 0.6 0.5 0.4 0.3

c-Met / GAPDH 0.2 * 0.1 0 Blank Mock ShC2 Blank Mock ShC2 Blank Mock ShC2 d

Blank Mock ShC2

Fig. 5 Depletion of c-Met inhibits the tumorigenicity of CNE-2 loading control. *P<0.05, vs. Mock. d TUNEL staining of the cells in vivo. a The tumor size curve of the xenografts formed from sections from xenografts (×200 magnification). Arrow indicated CNE-2 or its derived cells as indicated. b The morphology of the apoptotic cells (brown granules). Blank: untreated CNE-2 cells; xenografts formed from CNE-2 or its derived cells as indicated. c Mock: CNE-2 cells transfected with a scrambled shRNA; shC2: The expression level of c-Met in xenografts derived from the cells as CNE-2 cells transfected with c-Met shRNA2 indicated was determined by Western blot. GAPDH served as

important to overcome the potential resistance of NPC to c- the tumorigenicity of CNE-2 cells in vivo, which was at least Met inhibitors. partially dependent on the apoptosis induced by c-Met In conclusion, our present study have shown that shRNA- knockdown. Our data suggest that c-Met plays the key role mediated depletion of c-Met inhibited the proliferation, in the tumorigenesis of NPC and represents a novel migration, and invasion of CNE-2 cells in vitro, and reduced therapeutic target for NPC. Author's personal copy

1224 Tumor Biol. (2011) 32:1217–1224

Acknowledgments This study was supported by grants from the NADPH production leading to cell death. Oncogene. 2011;9: National Natural Science Foundation of China (no. 30801282), and 1127–34. the Research Fund for the Doctoral Program of Higher Education of 17. Zhou HY, Wan KF, Ip CK, Wong CK, Mak NK, Lo KW, et al. China (no. 20020487062). Hepatocyte growth factor enhances proteolysis and invasiveness of human nasopharyngeal cancer cells through activation of PI3K Conflicts of interest None and JNK. FEBS Letters. 2008;582:3415–22. 18. Maulik G, Shrikhande A, Kijima T, Ma PC, Morrison PT, Salgia R. Role of the hepatocyte growth factor receptor, c-Met, in References oncogenesis and potential for therapeutic inhibition. Cytokine Growth Factor Rev. 2002;1:41–59. 19. Grabellus F, Konik MJ, Worm K, Sheu SY, van de Nes JA, Bauer 1. Zhengrong Wu, Li G, Lirong Wu, Weng D, Li X, Yao K. Cripto-1 S, et al. MET overexpressing chordomas frequently exhibit overexpression is involved in the tumorigenesis of nasopharyngeal polysomy of chromosome 7 but no MET activation through carcinoma. BMC Cancer. 2009;3:1–14. sarcoma-specific gene fusions. Tumour Biol. 2010;31:157–63. 2. Spano JP, Busson P, Atlan D, Bourhis J, Pignon JP, Esteban C, et 20. Kuniyasu H, Yasui W, Kitadai Y, Yokozaki H, Ito H, Tahara E. al. Nasopharyngeal carcinoma: an update. Eur J Cancer. Frequent amplification of the c-met gene in scirrhous type 2003;15:2121–35. stomach cancer. Biochem Biophys Res Commun. 1992;189: 3. Farias TP, Dias FL, Lima RA, Kligerman J, de Sá GM, Barbosa 227–32. MM, et al. Prognostic factors and outcome for nasopharyngeal 21. Di Renzo MF, Olivero M, Serini G, Orlandi F, Pilotti S, Belfiore carcinoma. Arch Otolaryngol Head Neck Surg. 2003;7:794–9. A, et al. Overexpression of the c-MET/HGF receptor in human 4. Teo PM, Kwan WH, Lee WY, Leung SF, Johnson PJ. Prognos- thyroid carcinomas derived from the follicular epithelium. J ticators determining survival subsequent to distant metastasis from Endocrinol Invest. 1995;18:134–9. nasopharyngeal carcinoma. Cancer. 1996;12:2423–31. 22. Xie B, Xing R, Chen P, Gou Y, Li S, Xiao J, et al. Down- 5. Al-Sarraf M, LeBlanc M, Giri PG, Fu KK, Cooper J, Vuong T, regulation of c-Met expression inhibits human HCC cells growth et al. Chemoradiotherapy versus radiotherapy in patients with and invasion by RNA interference. J Surg Res. 2010;162:231–8. advanced nasopharyngeal cancer: phase III randomized Intergroup 23. Mariotti M, Castiglioni S, Maier JA. Inhibition of T24 human study 0099. J Clin Oncol. 1998;4:1310–7. bladder carcinoma cell migration by RNA interference suppressing 6. Ma BB, Chan AT. Recent perspectives in the role of chemother- the expression of HD-PTP. Cancer Lett. 2009;1:155–63. apy in the management of advanced nasopharyngeal carcinoma. 24. Su JL, Yang PC, Shih JY, Yang CY, Wei LH, Hsieh CY, et al. The Cancer. 2005;1:22–31. VEGF-C/Flt-4 axis promotes invasion and metastasis of cancer 7. Yu MC, Yuan JM. Epidemiology of nasopharyngeal carcinoma. cell. Cancer Cell. 2006;3:209–23. Semin Cancer Biol. 2002;6:421–9. 25. Cattaruzza S, Perris R. Proteoglycan control of cell movement 8. Burt RD, Vaughan TL, Nisperos B, Swanson M, Berwick M. A during wound healing and cancer spreading. Matrix Biol. prospective association between the HLA-A2 antigen and naso- 2005;6:400–17. pharyngeal carcinoma in US Caucasians. Int J Cancer. 26. Jeffers M, Fiscella M, Webb CP, Anver M, Koochekpour S, Vande 1994;4:465–7. Woude GF. The mutationally activated Met receptor mediates 9. Lo KW, Teo PM, Hui AB, To KF, Tsang YS, Chan SY, et al. High motility and metastasis. Proc Natl Acad Sci U S A. resolution allelotype of microdissected primary nasopharyngeal 1998;24:14417–22. carcinoma. Cancer Res. 2000;13:3348–53. 27. Kermorgant S, Aparicio T, Dessirier V, Lewin MJ, Lehy T. 10. Bottaro DP, Rubin JS, Faletto DL, Chan AM, Kmiecik TE, Vande Hepatocyte growth factor induces colonic cancer cell invasiveness Woude GF, et al. Identification of the hepatocyte growth factor via enhanced motility and protease overproduction. Evidence for PI3 receptor as the c-met proto-oncogene product. Science. kinase and PKC involvement. Carcinogenesis. 2001;7:1035–42. 1991;4995:802–4. 28. Gual P, Giordano S, Williams TA, Rocchi S, Van Obberghen E, 11. Comoglio PM, Giordano S, Trusolino L. Drug development of Comoglio PM. Sustained recruitment of phospholipase C-gamma MET inhibitors: targeting oncogene addiction and expedience. Nat to Gab1 is required for HGF-induced branching tubulogenesis. Rev Drug Discov. 2008;6:504–16. Oncogene. 2000;12:1509–18. 12. Di Renzo MF, Narsimhan RP, Olivero M, Bretti S, Giordano S, 29. Gual P, Giordano S, Anguissola S, Parker PJ, Comoglio PM. Medico E, et al. Expression of the Met/HGF receptor in normal Gab1 phosphorylation: a novel mechanism for negative regulation and neoplastic human tissues. Oncogene. 1991;11:1997–2003. of HGF receptor signaling. Oncogene. 2001;2:156–66. 13. Christensen JG, Burrows J, Salgia R. c-Met as a target for human 30. Tulasne D, Foveau B. The shadow of death on the MET tyrosine cancer and characterization of inhibitors for therapeutic intervention. kinase receptor. Cell Death Differ. 2008;3:427–34. Cancer Lett. 2005;1:1–26. 31. Ueda K, Iwahashi M, Matsuura I, Nakamori M, Nakamura M, 14. Xie LQ, Bian LJ, Li Z, Li Y, Li ZX, Li B. Altered expression of E- Ojima T, et al. Adenoviral-mediated gene transduction of the cadherin by hepatocyte growth factor and effect on the prognosis of hepatocyte growth factor (HGF) antagonist, NK4, suppresses nasopharyngeal carcinoma. Ann Surg Oncol. 2010;7:1927–36. peritoneal metastases of gastric cancer in nude mice. Eur J Cancer. 15. Jeffers M, Rong S, Woude GF. Hepatocyte growth factor/scatter 2004;14:2135–42. factor-Met signaling in tumorigenicity and invasion/metastasis. J 32. Li W, Cai S, Cai L, Li X. Anti-apoptotic effect of hepatocyte Mol Med. 1996;9:505–13. growth factor from actinomycin D in hepatocyte-derived HL7702 16. Lui VWY, Wong EYL, Ho K, Ng PKS, Lau CPY, Tsui SKW, et al. cells is associated with activation of PI3K/Akt signaling. Toxicol Inhibition of c-Met downregulates TIGAR expression and reduces Lett. 2006;2:142–8.