Expression of Neuropilin-2 in salivary adenoid cystic carcinoma: Its implication in tumor progression and angiogenesis

Pathology – Research and Practice 206 (2010) 793–799

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Original Article

Expression of Neuropilin-2 in salivary adenoid cystic carcinoma: Its implication in tumor progression and angiogenesis Yu Cai a,b,1 , Rong Wang b,1 , Yi-Fang Zhao a , Jun Jia a,∗ , Zhi-Jun Sun a , Xin-Ming Chen c a

Department of Oral and Maxillofacial Surgery, School and Hospital of Stomatology, Wuhan University, Wuhan 430079, China The State Key Laboratory Breeding Base of Basic Science of Stomatology (Hubei-MOST) & Key Laboratory of Oral Biomedical Engineering of Ministry of Education, School & Hospital of Stomatology, Wuhan University, Wuhan, China c Department of Oral Pathology, School and Hospital of Stomatology, Wuhan University, Wuhan, China b

a r t i c l e

i n f o

Article history: Received 15 March 2010 Received in revised form 1 August 2010 Accepted 4 August 2010 Keywords: Neuropilin-2 Salivary adenoid cystic carcinoma Tumor progression Angiogenesis

a b s t r a c t Neuropilin-2(Nrp2), which is a nontyrosine kinase transmembrane glycoprotein, can promote angiogenesis and is a poor prognostic factor in some human cancers. In the present study, to explore the expression and potential function of Nrp2 in salivary adenoid cystic carcinoma (SACC), immunohistochemistry was used to examine the Nrp2 expression in 50 SACCs and 20 normal salivary gland tissues nearby SACCs. The result showed that immunoreactivity for Nrp2 was detected in 47 of 50 SACCs, and its expression level had significant correlations with microvessel density, tumor size, TMN clinical stage, vascular invasion, and metastasis (P < 0.05) of SACCs. In addition, inhibition of Nrp2 function by the receptor–ligand interactionblocking antibody decreased cell migration, invasion, and angiogenic promotion without influences on the cell proliferation of Acc-3 cells. Taken together, the expression of Nrp2 protein is significantly correlated with tumor progression and angiogenesis in SACCs. These results suggest that Nrp2 may be a potential therapeutic target for SACCs. © 2010 Elsevier GmbH. All rights reserved.

Introduction Salivary adenoid cystic carcinoma (SACC) comprises approximately 10% of all epithelial salivary tumors and most frequently involves the parotid, submandibular, and minor salivary glands. Typically, SACC presents persistent slow growth, high rates of recurrence, perineural invasion, and distant metastasis [1]. It has a relentless clinical course and usually results in a very poor long-term outcome: only 20% of patients diagnosed with distant metastasis survive 5 years [2]. Although several investigators suggest that angiogenesis may be a possible mechanism for the invasiveness and aggressive metastatic dissemination of SACC, the precise mechanism responsible for its carcinogenesis has not been fully clarified [3]. Neuropilin-2 (Nrp2) is a nontyrosine kinase transmembrane glycoprotein with predicted molecular masses of 130–140 kDa [4], initially characterized as a receptor for the vascular endothelial growth factor (VEGF) and the semaphorin (SEMA) families, two unrelated ligand families involved in angiogenesis and neuronal guidance [5,6]. Although expression of Nrp2 was originally thought to be limited to neurons, this receptor has since been identified

∗ Corresponding author. Tel.: +86 27 87686215; fax: +86 27 87873260. E-mail address: [email protected] (J. Jia). 1 These two authors contributed to this work equally. 0344-0338/$ – see front matter © 2010 Elsevier GmbH. All rights reserved. doi:10.1016/j.prp.2010.08.001

on normal vascular and lymphatic endothelial cells [7,8], as well as on endothelial cells of hemangiomas [9]. Notably, a couple of very recent articles reported that the expression of Nrp2 is also ubiquitous in some tumor cells such as osteosarcoma, melanoma, nonsmall cell lung carcinoma, colorectal cancer, pancreatic ductal adenocarcinoma and breast cancer [10–16]. Thus, the idea of a specific function of Nrp2 in tumor has aroused increasing interest. Some researches have shown that Nrp2 expression in colorectal carcinoma cell and pancreatic ductal adenocarcinoma (PDAC) could enhance the abilities of cell survival, migration, invasion, in vivo tumor growth, and metastasis [12,16]. Using an antibody targeting Nrp2, Caunt et al. showed that inhibition of Nrp2 in a lung metastasis murine model prevented tumor metastases by blocking the formation of tumor-associated lymphatics [8]. Recently, studies by Yasuoka et al. showed that Nrp2 expression in breast cancer promoted lymph node metastasis via regulation of CXC chemokine receptor 4 expression [15]. Moreover, reduction in Nrp2 in PDAC cells led to decreased tumor-induced angiogenesis [12]. These studies indicated that Nrp2 expression in tumor cells is significantly correlated with increased angiogenesis, metastasis, and poor prognosis, which suggests that Nrp2 plays a critical role in tumor progression and may be a new molecular target for antitumor strategies. Nevertheless, the expression and function of Nrp2 in SACCs remain largely unknown. In this study, we examined Nrp2 expression in SACCs and adjacent uninvolved salivary gland. Furthermore,

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receptor–ligand interaction-blocking antibody to Nrp2 was used to assess the effects of Nrp2 on SACC cell proliferation, migration, invasion, and tumor-induced angiogenesis in vitro. Materials and methods Specimens Tissue samples of primary SACC and tumor-free salivary gland tissues around SACC were retrieved from archival material in the Department of Oral Pathology, School of Stomatology, Wuhan University, from 2000 to 2006. These samples were reviewed by two consultant pathologists before immunostaining both to confirm the diagnosis and to categorize SACCs into three histological patterns: cribriform, tubular, and solid according to the WHO classification [17]. Fifty SACCs (15 cribriform, 15 tubular, and 20 solid) and 20 normal salivary gland tissues that fulfilled the above criteria were employed. Clinical staging, performed according to the 2005 criteria of the WHO histological classification of tumors of the salivary glands, was evaluated by reviewing medical charts and pathological records.

score, when summed (intensity + extension), was 0 and the maximum was 7. The combined staining score (intensity + extension) ≤3 was considered as low staining; a score between 4 and 5 was considered as moderate staining; a score between 6 and 7 was considered strong staining. Furthermore, if staining for Nrp2 was partially moderate and partially mild, the weighted score was used for the evaluation of intensity, and the intensity was determined as follows: the weighted score for intensity = (the percentage of moderately stained cells in all positive tumor cells × 2) + (the percentage of mildly stained cells in all positive tumor cells × 1). Therefore, the combined staining score was equal to the weighted score for intensity adding the score for extension. MVD was evaluated according to the method described previously [19]. The entire tumor section was scanned at low magnification (100×) to find the area that showed the most intense neovascularization, i.e., the highest density of brown-stained, CD34-positive cells (hotspot). Five most highly vascularized hotspots in each case were selected in a 400× field. Any immunoreactive endothelial cell or endothelial cell cluster which was clearly separated from adjacent microvessels was considered as a single countable microvessel. Vessel lumens, although usually present, were not necessary for a structure to be defined as a microvessel.

Immunohistochemistry Cell culture and treatments The immunohistochemical analysis was performed as described by us previously [18]. Briefly, tissue samples embedded in paraffin were serially cut into 4-␮m sections, deparaffinized, and antigenretrieved using a 600 W microwave oven. After that, the sections were washed with phosphate-buffered saline (PBS) and incubated with 3% hydrogen peroxide and 10% goat or rabbit serum for 15 min, followed by an overnight incubation with polyclonal goat antihuman Nrp2 (AF2215, R&D Systems, Minneapolis, MN, USA. This antibody can exclusively recognize the human Nrp2 extracellular domain, and the subtype of immunoglobulin is goat IgG.) at a dilution of 1:400, CD34 monoclonal antibody (Dako, Carpinteria, CA, USA) at a dilution of 1:500. Then, antibody binding was detected by horseradish peroxidase-conjugated secondary antibody using diaminobenzidine substrate kit (Dako) according to the manufacturer’s protocol. Negative immunohistochemical controls consisted of tissue sections from each specimen, processed in parallel with omission of the primary antibodies. Known positive tissues for each marker were used as positive controls, while the vascular endothelium, which exhibits a consistently strong, predominantly cell membrane staining for Nrp2, served as an internal positive control.

The cell lines Acc-2, Acc-M, and Acc-3[20] were grown in DMEM media (Hyclone, Logan, UT, USA) supplemented with 10% fetal bovine serum (Hyclone) and maintained at 37 ◦ C under an atmosphere of 5% CO2 . The human umbilical vein endothelial cell line HUV-EC-C (ATCC CRL 1730) was maintained in DMEM media supplemented with 10% fetal bovine serum and 0.03 mg/ml endothelial cell growth supplement (ECGS) in a humidified 5% CO2 atmosphere. To inhibit the function of Nrp2 in Acc-3, receptor–ligand interaction-blocking antibody to Nrp2 (AF2215, R&D) was used. Human Acc-3 cells were seeded at a density of 5 × 105 per well in 6-well plates. Confluent cells were serum-starved (FBS was eliminated in all the following experiments) for 24 h, then treated with 5 ␮g/ml (according to the manufacturer’s suggestions) Nrp2 antibody for 72 h. To obtain the conditioned medium (CM), sub-confluent Acc-3 cells were serum-starved for 24 h in 6 well plates. Then, the cells were treated for 72 h with or without 5 ␮g/ml Nrp2 antibody, after which the medium was replaced with 2.0 ml serum-free DMEM medium to eliminate the Nrp2 antibody. After incubation for 24 h, the CM were obtained, stored at −20 ◦ C, and used for the following experiments.

Evaluation of staining RNA isolation, cDNA synthesis, and RT-PCR assay Sections were blindly evaluated by two investigators (Y.C. and R.W.) in an effort to provide a consensus on staining patterns using light microscopy (Olympus, Tokyo, Japan). Nrp2 staining was localized within the membrane, and assessed according to the methods described previously [2] with minor modifications. Each case was rated according to a score that added a staining intensity scale (viewed at a magnification of 200×) to the area of staining seen (at a magnification of 40×). The intensity of Nrp2 staining in the tumor cells was judged by that in the endothelial cells in the same tissue specimen on the following scale: 0, no staining; 1+, mild staining (less intensity than the Nrp2 present in blood vessels in the same tissue section); 2+, moderate staining (intensity is equal to Nrp2 present in blood vessels); 3+, intense staining (intensity is stronger than the Nrp2 present in blood vessels). The area of staining (extension) was evaluated as follows: 0, no staining of cells in any microscopic field; 1+, <25% of tissue stained positive; 2+, between 25% and 50% stained positive; 3+, between 50% and 75% stained positive; 4+, >75% stained positive. The minimum

Total RNA from Acc-2, Acc-M, and Acc-3 cells was isolated using TRIZOL reagent (Invitrogen, Burlington, ON, USA) according to the manufacturer’s instructions. RNA (1.0 ␮g) was used as template for the synthesis of cDNA (20 ␮l) with OligodT and AMV reverse transcriptase (Takara, Japan). One-fifth of the cDNA was PCR-amplified utilizing Taq polymerase (Takara) and specific primers. The primers used for RT-PCR were as follows: 5 -GTGGTTCATCTTGACCTTGT-3 for Nrp2; 5 and 5 -ATTCTTCTTCTGCAACCTCA-3 CCTGGCACCCAGCACAAT-3 and 5 -GCTGATCCACATCTGCTGGAA-3 for Beta-actin. The PCR products were electrophoresed in 2% agarose gel and stained with ethidium bromide. In vitro proliferative assays and cell cycle analysis For proliferative assay, tumor cells were seeded into 96-well plastic culture plates (103 cells/well) and allowed to adhere for 24 h. The media were then replaced with medium with 2% serum

Y. Cai et al. / Pathology – Research and Practice 206 (2010) 793–799

or serum-free DMEM medium. After 24 h, cells were treated with 5 ␮g/ml Nrp2 antibody. After 3 days and 6 days of incubation, cell viability was assayed by MTT (thiazolyl blue tetrazolium bromide) assay [21]. For cell cycle analysis, tumor cells were plated at a density of 2 × 105 per well in 6-well plates. After a 24-h attachment period, the media were changed to 2% serum or serum-free DMEM medium, and cells were maintained for 24 h and then treated with 5 ␮g/ml Nrp2 antibody. After 72 h, both adherent and detached cells were harvested, washed with PBS, and resuspended in 50 ␮g/ml propidium iodide (Sigma, St. Louis, MO, USA) for 30 min at room temperature in the dark. Flow cytometric analysis was done to examine the cell cycle. Negative control cells were treated with medium alone. Tumor cell migration and invasion assays Migration assays were performed in a standard 24-well Boyden chamber (Corning, Cambridge, MA, USA) by a modification of the method described by Albini et al. [22]. Briefly, 105 cells (treated with or without antibody for 72 h) in 100 ␮l DMEM was added to each insert, and 500 ␮l of DMEM containing 10% FBS was added to the well underneath the insert. After 72 h of incubation, the inner side of the insert was wiped with a wet swab to remove the cells while the outer side of the insert was gently rinsed with PBS and stained with 0.25% crystal violet for 10 min, rinsed again, and then allowed to dry. The inserts were then viewed under the microscope at 400× magnification. Migrated cells were counted in five random fields, and the assays were performed in quadruplicate. Invasion assays were done using a similar protocol with minor modifications. The inserts used in the invasion assays were coated with 1 mg/ml Matrigel (Sigma) and prehydrated with 1% FBSsupplemented medium for 30 min before the addition of the cell suspension. Invasion chambers were incubated for 72 h, and the numbers of invading cells were quantified. Endothelial cell migration assay Migration assays for ECs were performed according to the method described by Yamaguchi et al. [23]. Eight micron Nucleopore polyvinylpyrrolidine-free polycarbonate filters (Corning) were coated with 100 ␮g/ml of collagen type 1 (Amresco, Solon, OH, USA) in 0.2N acetic acid for 2 days and air-dried. The filter was placed over a bottom chamber containing the 0.6 ml CM from Acc-3 treated with or without 5 ␮g/ml Nrp2 antibody. HUV-EC-C cells were suspended in DMEM, and 10,000 cells in 50 ␮l were added to each well in the upper chamber. The assembled chemotaxis chamber was incubated for 24 h at 37 ◦ C with 5% CO2 to allow cells to migrate through the collagen-coated polycarbonate filter. Non-migrated cells on the upper surface of the filter were removed, and the filter was stained with crystal violet. Migrated cells were counted in five random high-power microscopic fields (400×), and the assays were performed in quadruplicate. Tube formation assay 500 ␮l type I collagen gel solution (0.3%), 100 ␮l 10× DMEM and 400 ␮l NaOH-Heppers buffer were mixed in an ice cold condition and pipetted into a 6-well plate and kept for 30 min at 37 ◦ C. HUV-EC-C cells were seeded into the layer of the gel at a density of 5 × 104 cells/well with 10% FBS medium. After 24 h, the medium was replaced by 0.8 ml CM from Acc-3 treated with or without 5 ␮g/ml Nrp2 antibody, which was added with 0.2 ml 10% FBS DMEM medium to reach the 2% FBS-CM final concentration.

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The 1.0 ml 2% FBS medium was used as the control. After 3 days, the tubular structures organized and gradually elongated and formed networks by HUV-EC-C cells. The number of tubular-like structures in five random high-power microscopic fields (400×) was counted. Statistical analysis Statistical analysis was done using SPSS (version 13.0). The Spearman rank correlation coefficient test and linear tendency test were applied for the correlation between the expression of Nrp2 and MVD. The association between Nrp2 expression and clinicopathological factors was analyzed by the chi-squared test. All in vitro experiments were repeated at least three times. The Student’s t-test was used to evaluate the differences between experimental and control groups. Significance was defined at the level of P < 0.05. Results Expression of Nrp2 in normal salivary gland and SACCs Immunoreaction for Nrp2 was detected in the SACC cells (Fig. 1B–E). The Nrp2-specific proteins showed moderate to strong staining in 45 of 50 (90%) SACC specimens, and the mean score was 4.31 ± 1.832 (mean ± SD). The membrane of tubular cells in normal salivary glands showed a slight staining of Nrp2, but staining intensity was significantly lower than in SACC tumor cells (P < 0.01), whereas there was no staining in the acinar cells of normal salivary glands (Fig. 1A and B). When RNA from 3 commonly used SACC cell lines (Acc-2, Acc-3 and Acc-M) was analyzed for Nrp2 expression, it was detected in all the cells (Fig. 1F). Relationship between Nrp2 expression and MVD in SACCs To evaluate whether there was any link between MVD and Nrp2 expression, the Spearman correlation analysis was made to quantitate the degree of the linear association between two variables. The expression levels of Nrp2 were significantly correlated to MVD in SACCs (Fig. 2, P < 0.01). Relationship between Nrp2 expression and clinicopathological parameters of SACCs Table 1 shows that Nrp2 expression was significantly correlated with some of the clinicopathological factors examined, such as tumor size, TNM clinical stage, vascular invasion, and metastasis (P < 0.05). Furthermore, among the three histological types of SACC, the expression of Nrp2 was significantly higher in the solid type than that in cribriform and tubular types (P < 0.05), whereas no significant difference was found between the cribriform and tubular types (Table 1). Anti-Nrp2 antibody inhibited migration and invasion of tumor cells As the neuropilins are co-receptors for VEGFRs in both tumor cells and endothelial cells, we determined the effect of Nrp2 expression on Acc-3 cells, which have been demonstrated to express VEGF and VEGFRs [24] by inhibiting the function of Nrp2 using the receptor–ligand interaction-blocking antibody. MTT assay (data not shown) and flow cytometric analysis were applied to determine the effect of Nrp2 on the in vitro proliferation rate. The Acc-3 cells treated with or without anti-Nrp2 antibody showed a similar in vitro proliferation rate up to 72 h after plating in 2% serum or serum-free DMEM medium (Table 2, P > 0.05).

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Fig. 1. Nrp2 immunoreactivity in specimens (magnification 400×). Nrp2 was absent in normal salivary gland (A) but was expressed in cribriform (C), tubular (D), and solid (E) patterns of SACC. In the gland-invasion type (B), Nrp2 protein is immunonegative in the normal salivary gland, but positive in the invasive SACC cells. Furthermore, PCR assay showed that mRNA of Nrp2 was present in all SACC cell lines (Acc-2, Acc-3 and Acc-M).

To evaluate the effect of Nrp2 on in vitro migration and invasion, a standard Boyden chamber assay was used. Acc-3 cells treated with anti-Nrp2 antibody showed a 51% decrease in the ability to migrate (Fig. 3A, P < 0.05) and a 50% decrease in the ability to invade through a matrigel-coated membrane (Fig. 3B, P < 0.05).

Anti-Nrp2 antibody inhibited tumor-induced angiogenesis The ability of tumor-induced angiogenesis was evaluated by the in vitro endothelial cell migration and tube formation assay. In the presence of medium from Acc-3, HUV-EC-Cs formed organized elongated tube-like structures resembling capillaries with an extensive network, while in the presence of medium from Acc3 treated with the Nrp2 antibodies, the migration of ECs and the formation of the tubular-like structures decreased (Fig. 3C and D, P < 0.05).

Discussion

Fig. 2. Correlation between Nrp2 expression and MVD. Spearman correlation and linear regression were used to determine the relationship between Nrp2 expression and MVD. MVD correlated positively with Nrp2 expression.

SACC is characterized by local recurrence, late distant metastasis, and poor response to conventional chemotherapy. Therefore, new therapeutic approaches, including molecular targeted therapies, are applied for the benefit of SACC patients. Current molecular targets in SACCs include c-KIT, EGFR, the human epidermal growth receptor-2, and estrogen and progesterone receptor; however, the effectiveness is not inspiring [24]. Therefore, new molecular targeted therapies directed towards additional and/or alternate molecular targets are urgently needed. Based on the positive expression of Nrp2 in many types of cancers and its possible role in carcinogenesis, we detected Nrp2 in 50 cases of SACCs and in 20

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Table 1 Correlation between Nrp2 expression and clinicopathological features. n (%)

Nrp2

P

0–3 Age <50 ≥50 Gender Male Female Size ≤2 cm >2 cm Site Major Minor TNM stage T1 + T2 T3 + T4 Perineural invasion Negative Positive Vascular invasion Negative Positive Metastasis Negative Positive Histotypes Cribriform Tubular Solid *

4–5

6–7

26 (52%) 24 (48%)

7 7

11 10

8 7

0.862

18 (36%) 32 (64%)

3 11

8 13

7 8

0.165

16 (32%) 34 (68%)

9 5

5 16

2 13

0.03*

12 (24%) 38 (76%)

3 11

6 15

3 12

0.919

31 (62%) 19 (38%)

12 2

13 8

6 9

0.011*

24 (48%) 26 (52%)

6 8

10 11

8 7

0.581

35 (70%) 15 (30%)

13 1

16 5

6 9

0.001*

36 (72%) 14 (28%)

12 2

17 4

7 8

0.018*

23 (46%) 17 (34%) 10 (20%)

8 5 1

11 8 2

4 4 7

0.622 (C vs T) 0.007* (C vs S) 0.027* (T vs S)

P < 0.05 by ANOVA test.

cases of normal salivary glands. We report here for the first time that Nrp2 is strongly expressed in SACCs, and its expression level is correlated with MVD and the malignant degree of SACCs. The roles of Nrp2 in cancer biology might be attributable to their mediating the migratory and invasive activity of tumor cells. Studies from in vitro systems have demonstrated that reduced Nrp2 expression in PDAC and colorectal carcinoma cells leads to decreased migration, invasion, and in vivo tumor growth [12]. In addition, inhibition of the function of Nrp2 blocked vascular and lymphatic endothelial cell (EC) migration [7,8]. Nevertheless, in all these cells, there is no relationship between the expression of Nrp2 and cell proliferation. To further understand the mechanisms of Nrp2 expression in SACCs, cell proliferation, migration, and invasion were examined when the function of Nrp2 was inhibited by the Nrp2 antibody. Similar to what has been previously reported in human PDACs, colorectal carcinoma cells, and ECs, inhibition of the Nrp2 function decreased cell migration and invasion, but there was no influence on the cell proliferation of SACC cells. Angiogenesis or the formation of new blood vessels is an essential component in tumor cell survival, which promulgates tumor metastasis. Newly formed blood vessels provide an increased availability of essential oxygen and nutrients to the burgeoning tumor to permit expansion beyond an avascular mass of 1–2 mm3 [25]. Previous studies have demonstrated that Nrp2 expression in cancer

cells was significantly correlated with angiogenesis [12,14], and, to some degree, our recent research also agrees with those pieces of evidence that the protein level of Nrp2 was significantly higher in SACCs than in normal salivary gland, and there was a positive correlation with MVD in SACCs. To further investigate the angiogenesis abilities of Nrp2 expression in SACCs, the in vitro angiogenesis model was also employed. Through in vitro ECs migration assay and tube formation assay, our present study shows that inhibition of the Nrp2 function in Acc-3 cells significantly decreased the migration and tube formation of HUV-EC-C cells. Regarding the question of how Nrp2 functions in the process of carcinogenesis and tumor-induced angiogenesis, it has been widely accepted that Nrp2, which has short intracellular domains that are not known to have any enzymatic or signaling activity [5], functions as an obligate co-receptor by cooperatively enhancing the activity of the VEGF kinase receptors. Gray et al. [16] showed that decreased Nrp2 expression in HCT-116 colorectal cancer cells led to substantial reductions in the phosphorylation of Akt and to downstream antiapoptosis protein BAD by inhibiting activation of VEGFR-1. Dallas et al. [12] also pointed out that Nrp2 promoted the activation of Akt through VEGFR single pathway in BxPC3 pancreatic cancer cells. In addition, it has been demonstrated that Nrp2 promotes tumor angiogenesis by up-regulating the constitutive expression of the angiogenic mediator Jagged-1 in BxPC3 [12]. Recently, Yasuoka et al. [15] indicated that the expression of CXC chemokine recep-

Table 2 Cell cycle distribution of Acc-3 treated with or without anti-Nrp2 antibody after 72 h under 2% serum and serum-free DMEM medium. Phase of cell cycle

G0 /G1 S G2 /M

DMEM with 2% serum

P

Normal

Anti-Nrp2

80.98 ± 1.35 11.07 ± 1.01 7.95 ± 0.37

80.75 ± 1.76 11.08 ± 1.00 8.18 ± 0.22

Normal group vs anti-Nrp2 group, P > 0.05 by t-test, n = 4.

0.807 0.471 0.165

Serum-free DMEM medium

P

Normal

Anti-Nrp2

79.58 ± 1.42 12.83 ± 1.63 7.60 ± 0.42

80.6 ± 1.26 12.00 ± 0.96 7.40 ± 0.41

0.885 0.663 0.718

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Fig. 3. The effect of Nrp2 on SACC cell lines in vitro. Anti-Nrp2 antibody inhibited migration (A) and invasion (B) of tumor cells; in the presence of medium from Acc-3 treated with Nrp2 antibody, the migrating cells (C) and the formation of the tubular-like structures (D) were decreased. HPF, high-power microscopic field (magnification 400×).

tor 4 was also regulated by Nrp2 dependent on VEGFR signaling. All these pieces of evidence indicate that the expression of Nrp2 in cancer cells is also associated with the activation of VEGFR. Similar to most cancers, VEGFR signaling was excessively activated in SACCs, while the inhibition of VEGFR pathway has significant antitumor and antiangiogenic effects on SACC cells [24,26]. These results suggest that Nrp promotes the progression of SACCs also probably through the activation of VEGFR signaling pathway. However, Caunt and Pan [8,27] have suggested an alternative possibility that Nrp2 might modulate ECs migration, angiogenesis, and lymphangiogenesis by a mechanism other than enhancing VEGFR activation. Therefore, our further investigations should focus on the exact role of Nrp2 in the regulation of tumor progression and angiogenesis in SACCs. Taken together, the expression of Nrp2 protein is significantly correlated with tumor progression and angiogenesis in SACCs. Inhibition of the function of Nrp2 in Acc-3 cells led to significant reductions in migration, invasion, and tumor cell-induced angiogenesis. These results suggest that Nrp2 may be a potential therapeutic target for SACCs. Acknowledgement This work was funded by grant NSFC 30801305 from National Natural Science Foundation of China to Jun Jia.

References [1] J.F. He, M.H. Ge, X. Zhu, C. Chen, Z. Tan, Y.N. Li, Z.Y. Gu, Expression of RUNX3 in salivary adenoid cystic carcinoma: implications for tumor progression and prognosis, Cancer Sci. 99 (2008) 1334–1340. [2] J. Zhang, B. Peng, X. Chen, Expressions of nuclear factor kappaB, inducible nitric oxide synthase, and vascular endothelial growth factor in adenoid cystic carcinoma of salivary glands: correlations with the angiogenesis and clinical outcome, Clin. Cancer Res. 11 (2005) 7334–7343. [3] H. Ishibashi, T. Shiratuchi, K. Nakagawa, M. Onimaru, T. Sugiura, K. Sueishi, K. Shirasuna, Hypoxia-induced angiogenesis of cultured human salivary gland carcinoma cells enhances vascular endothelial growth factor production and basic fibroblast growth factor release, Oral Oncol. 37 (2001) 77–83. [4] C.A. Staton, I. Kumar, M.W. Reed, N.J. Brown, Neuropilins in physiological and pathological angiogenesis, J. Pathol. 212 (2007) 237–248. [5] H. Chen, A. Chedotal, Z. He, C.S. Goodman, M. Tessier-Lavigne, Neuropilin2, a novel member of the neuropilin family, is a high affinity receptor for the semaphorins Sema E and Sema IV but not Sema III, Neuron 19 (1997) 547–559. [6] C. Pellet-Many, P. Frankel, H. Jia, I. Zachary, Neuropilins: structure, function and role in disease, Biochem. J. 411 (2008) 211–226. [7] B. Favier, A. Alam, P. Barron, J. Bonnin, P. Laboudie, P. Fons, M. Mandron, J.P. Herault, G. Neufeld, P. Savi, J.M. Herbert, F. Bono, Neuropilin-2 interacts with VEGFR-2 and VEGFR-3 and promotes human endothelial cell survival and migration, Blood 108 (2006) 1243–1250. [8] M. Caunt, J. Mak, W.C. Liang, S. Stawicki, Q. Pan, R.K. Tong, J. Kowalski, C. Ho, H.B. Reslan, J. Ross, L. Berry, I. Kasman, C. Zlot, Z. Cheng, J. Le Couter, E.H. Filvaroff, G. Plowman, F. Peale, D. French, R. Carano, A.W. Koch, Y. Wu, R.J. Watts, M. TessierLavigne, A. Bagri, Blocking neuropilin-2 function inhibits tumor cell metastasis, Cancer Cell 13 (2008) 331–342. [9] M.L. Calicchio, T. Collins, H.P. Kozakewich, Identification of signaling systems in proliferating and involuting phase infantile hemangiomas by genome-wide transcriptional profiling, Am. J. Pathol. 174 (2009) 1638–1649.

Y. Cai et al. / Pathology – Research and Practice 206 (2010) 793–799 [10] P.M. Lacal, C.M. Failla, E. Pagani, T. Odorisio, C. Schietroma, S. Falcinelli, G. Zambruno, S. D’Atri, Human melanoma cells secrete and respond to placenta growth factor and vascular endothelial growth factor, J. Invest. Dermatol. 115 (2000) 1000–1007. [11] T. Kawakami, T. Tokunaga, H. Hatanaka, H. Kijima, H. Yamazaki, Y. Abe, Y. Osamura, H. Inoue, Y. Ueyama, M. Nakamura, Neuropilin 1 and neuropilin 2 co-expression is significantly correlated with increased vascularity and poor prognosis in nonsmall cell lung carcinoma, Cancer 95 (2002) 2196–2201. [12] N.A. Dallas, M.J. Gray, L. Xia, F. Fan, G. van Buren 2nd, P. Gaur, S. Samuel, S.J. Lim, T. Arumugam, V. Ramachandran, H. Wang, L.M. Ellis, Neuropilin-2-mediated tumor growth and angiogenesis in pancreatic adenocarcinoma, Clin. Cancer Res. 14 (2008) 8052–8060. [13] N.A. Dallas, F. Fan, M.J. Gray, G. van Buren 2nd, S.J. Lim, L. Xia, L.M. Ellis, Functional significance of vascular endothelial growth factor receptors on gastrointestinal cancer cells, Cancer Metastasis Rev. 26 (2007) 433–441. [14] A. Handa, T. Tokunaga, T. Tsuchida, Y.H. Lee, H. Kijima, H. Yamazaki, Y. Ueyama, H. Fukuda, M. Nakamura, Neuropilin-2 expression affects the increased vascularization and is a prognostic factor in osteosarcoma, Int. J. Oncol. 17 (2000) 291–295. [15] H. Yasuoka, R. Kodama, M. Tsujimoto, K. Yoshidome, H. Akamatsu, M. Nakahara, M. Inagaki, T. Sanke, Y. Nakamura, Neuropilin-2 expression in breast cancer: correlation with lymph node metastasis, poor prognosis, and regulation of CXCR4 expression, BMC Cancer 9 (2009) 220. [16] M.J. Gray, G. Van Buren, N.A. Dallas, L. Xia, X. Wang, A.D. Yang, R.J. Somcio, Y.G. Lin, S. Lim, F. Fan, L.S. Mangala, T. Arumugam, C.D. Logsdon, G. Lopez-Berestein, A.K. Sood, L.M. Ellis, Therapeutic targeting of neuropilin-2 on colorectal carcinoma cells implanted in the murine liver, J. Natl. Cancer Inst. 100 (2008) 109–120. [17] G. Carlinfante, M. Lazzaretti, S. Ferrari, B. Bianchi, P. Crafa, P53, bcl-2 and Ki-67 expression in adenoid cystic carcinoma of the palate. A clinico-pathologic study of 21 cases with long-term follow-up, Pathol. Res. Pract. 200 (2005) 791–799. [18] J. Jia, Y. Bai, K. Fu, Z.J. Sun, X.M. Chen, Y.F. Zhao, Expression of allograft inflammatory factor-1 and CD68 in haemangioma: implication in the progression of haemangioma, Br. J. Dermatol. 159 (2008) 811–819.

799

[19] S. Rasheed, A.L. Harris, P.P. Tekkis, H. Turley, A. Silver, P.J. McDonald, I.C. Talbot, R. Glynne-Jones, J.M. Northover, T. Guenther, Assessment of microvessel density and carbonic anhydrase-9 (CA-9) expression in rectal cancer, Pathol. Res. Pract. 205 (2009) 1–9. [20] X.F. Guan, W.L. Qiu, R.G. He, X.J. Zhou, Selection of adenoid cystic carcinoma cell clone highly metastatic to the lung: an experimental study, Int. J. Oral Maxillofac. Surg. 26 (1997) 116–119. [21] T. Mosmann, Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays, J. Immunol. Methods 65 (1983) 55–63. [22] A. Albini, Y. Iwamoto, H.K. Kleinman, G.R. Martin, S.A. Aaronson, J.M. Kozlowski, R.N. McEwan, A rapid in vitro assay for quantitating the invasive potential of tumor cells, Cancer Res. 47 (1987) 3239–3245. [23] N. Yamaguchi, B. Anand-Apte, M. Lee, T. Sasaki, N. Fukai, R. Shapiro, I. Que, C. Lowik, R. Timpl, B.R. Olsen, Endostatin inhibits VEGF-induced endothelial cell migration and tumor growth independently of zinc binding, Embo J. 18 (1999) 4414–4423. [24] S. Choi, D. Sano, M. Cheung, M. Zhao, S.A. Jasser, A.J. Ryan, L. Mao, W.T. Chen, A.K. El-Naggar, J.N. Myers, Vandetanib inhibits growth of adenoid cystic carcinoma in an orthotopic nude mouse model, Clin. Cancer Res. 14 (2008) 5081– 5089. [25] J. Folkman, Angiogenesis and apoptosis, Semin. Cancer Biol. 13 (2003) 159–167. [26] M.N. Younes, Y.W. Park, Y.D. Yazici, M. Gu, A.A. Santillan, X. Nong, S. Kim, S.A. Jasser, A.K. El-Naggar, J.N. Myers, Concomitant inhibition of epidermal growth factor and vascular endothelial growth factor receptor tyrosine kinases reduces growth and metastasis of human salivary adenoid cystic carcinoma in an orthotopic nude mouse model, Mol. Cancer Ther. 5 (2006) 2696– 2705. [27] Q. Pan, Y. Chanthery, W.C. Liang, S. Stawicki, J. Mak, N. Rathore, R.K. Tong, J. Kowalski, S.F. Yee, G. Pacheco, S. Ross, Z. Cheng, J. Le Couter, G. Plowman, F. Peale, A.W. Koch, Y. Wu, A. Bagri, M. Tessier-Lavigne, R.J. Watts, Blocking neuropilin-1 function has an additive effect with anti-VEGF to inhibit tumor growth, Cancer Cell 11 (2007) 53–67.