Oral Oncology 50 (2014) 809–817
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Oral Oncology journal homepage: www.elsevier.com/locate/oraloncology
Loss of VHL expression contributes to epithelial–mesenchymal transition in oral squamous cell carcinoma Shu Zhang a,b,1, Xuan Zhou a,b,1, Bo Wang a,b, Kailiang Zhang c, Su Liu a,b, Kai Yue a,b, Lun Zhang a,b, Xudong Wang a,b,⇑ a b c
Department of Maxillofacial and E.N.T Oncology, Tianjin Medical University Cancer Institute & Hospital, Tianjin 300060, China National Clinical Research Center for Cancer, Key Laboratory of Cancer Prevention and Therapy, Tianjin 300060, China Department of Neurosurgery, Tianjin Medical University General Hospital, Laboratory of Neuro-oncology, Tianjin Neurological Institute, Tianjin 300052, China
a r t i c l e
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Article history: Received 25 January 2014 Received in revised form 18 May 2014 Accepted 6 June 2014 Available online 2 July 2014 Keywords: VHL Oral squamous cell carcinoma Epithelial–mesenchymal transition b-Catenin
s u m m a r y Objective: Loss of Von Hippel–Lindau (VHL) gene expression has been implicated in the development of human cancers. However, its function in oral squamous cell carcinoma (OSCC) remains undefined. The aim of this study was to clarify the VHL expression in OSCC and to explore the underlying mechanisms of VHL in modulating the epithelial–mesenchymal transition (EMT) in OSCC. Materials and methods: The expression of VHL, HIF-1a and EMT related proteins in OSCC tissues were evaluated by immunohistochemistry. The correlation of VHL with clinico-pathological characteristics, prognosis and EMT related proteins were analyzed. The roles of VHL on the cell morphology, proliferation, migration, and invasion were determined by MTT, scratch and transwell invasion assay in Tscca and Tca8113P160 cells. The EMT related proteins were determined by Western blot and immunofluorescence (IF) methods. Results: Loss of VHL expression was closely associated with pathologic grading, lymph node metastasis, poor prognosis, and EMT in OSCC. After re-expression of VHL, there was a cell morphologic change and motivation, proliferation, invasion of the cells were inhibited. The expression of Snail, N-cadherin and MMP-2/9, HIF-1a and VEGF were down-regulated in both the cell lines after transfection with VHL plasmid, while E-cadherin was up-regulated. Moreover, the effect of VHL suppressing b-catenin accumulation in nucleus was proved by Western blot and IF. Conclusion: VHL was significantly correlated with EMT process of OSCC. b-Catenin was an important downstream gene of VHL besides HIF-1a, which could induce the EMT process in OSCC. Ó 2014 Elsevier Ltd. All rights reserved.
Introduction Oral squamous cell carcinoma (OSCC) is a common type of malignant tumor in head and neck, with approximately 275,000 new cases worldwide each year [1]. It is characterized by a high degree of local invasiveness and a high rate of metastasis to the cervical lymph nodes [2]. Despite combined therapies, such as radical surgery, radiotherapy and neo-adjuvant chemotherapy, were used, the poor prognosis has not been significantly improved during the last decade. The 5-year overall survival rate is below 50% and is even lower in locally advanced or metastatic diseases ⇑ Corresponding author at: Department of Maxillofacial and E.N.T Oncology, Tianjin Medical University Cancer Institute & Hospital, Tianjin 300060, China. Tel./fax: +86 22 23340123. E-mail address: [email protected] (X. Wang). 1 These authors made equal contribution to this work. http://dx.doi.org/10.1016/j.oraloncology.2014.06.007 1368-8375/Ó 2014 Elsevier Ltd. All rights reserved.
[3,4]. Thus, it is urgent to promote the studies of the carcinogenesis and mechanism of metastasis in OSCC. It has been shown in our and others’ past researches that the process of epithelial–mesenchymal transition (EMT) is a key step for OSCC progression and metastasis [5–7]. Tumor cells undergoing EMT remodel from the epithelial phenotype to mesenchymal phenotype, accompanied by decrease in E-cadherin and increase in Snail, N-cadherin, vimentin and Twist, etc. [8]. These representative changes would facilitate cell losing their adhesiveness, dissolving the extracellular matrix that restrains them and spreading to the surrounding tissue. Moreover, the aforementioned typical markers of EMT are predictive of the patients’ survival [9]. The Wnt/b-catenin is one of the major signaling pathways in EMT. It was shown in a recent study that aberrant accumulated b-catenin in the cytoplasm is translocated to the nucleus where it binds to the Tcf/Lef, leading to the up-regulation of target genes, thus induce EMT in OSCC [10].
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The Von Hippel–Lindau gene (VHL) is the causal gene for VHL disease, which is a hereditary neoplastic syndrome, characterized by susceptibility for hyper-vascularized clear-cell renal cell carcinoma (ccRCC) [11]. Aberration in function of VHL gene, either through mutation, loss of heterozygosity (LOH), or methylation of VHL gene drives tumor initiation and progression [12,13]. It has been commonly known that VHL encoding product, VHL protein, acts as the recognition subunit of a multi-protein complex with ubiquitin E3 ligase activity, which targets the hypoxia inducible factor (HIF) for polyubiquitination and subsequent proteasomal degradation. Inactivation of VHL leads to HIF stabilization and activation that plays an important role in tumorigenesis [14]. Although evidence of abnormality in VHL inducing OSCC is evolving [15], little has been uncovered regarding the connection between VHL and EMT in OSCC. Based on earlier evidence that VHL gene deletion can activate b-catenin [16], an important mediator of EMT, we tested whether b-catenin played mediating role in loss of VHL regulated OSCC EMT process. In the study, we determined the expression of VHL, its target protein, HIF-1a, and EMT related proteins in the tissue samples of OSCC using immunohistochemistry (IHC), and attempted to elucidate the relationship between VHL and EMT in OSCC. Thus, we provided a novel mechanism of b-catenin that was regulated by VHL which could induce EMT in OSCC.
node status, were obtained from the medical records, as shown in Table 1. Survival information of the 80 patients was obtained from the visits by letters or by calls. Informed consent was obtained from all subjects. Immunohistochemistry (IHC) For IHC staining, paraffin-embedded tissue slides (4 lm thick) were deparaffinized, rehydrated and incubated with primary antibodies overnight at 4 °C. The following antibodies were used: VHL, HIF-1a, VEGF (Santa Cruz, CA, USA), Snail, N-cadherin, E-cadherin, matrix metalloproteina-2/9(MMP-2/9,Abcam, Cambridge, MA, USA), and b-catenin (Abmart, Shanghai, China). Then, they were incubated with biotin-labeled secondary antibody (Maxin Bio Corp., Fujian, China) for 1 h at room temperature and incubated with diaminobenzidine (Zhongshan Bio Corp., Beijing, China), counterstained with hematoxylin. Slides were dehydrated with different concentrations of alcohol and soaked in xylene, and then mounted with neutral balsam and visualized using light microscope. Ten representative fields at 400 magnification per slide were observed. The results were assessed by measuring both the staining intensity and the number of positive cells. The intensity of the positive reaction was scored, i.e. 0 = negative, 1 = light staining, 2 = moderate staining, and 3 = intense staining. Additionally, staining was scored on a scale of 0–3 according to the percentage of the cells involved: i.e. scale 0 means 0–5% positive cells; 1, 6–25% positive cells; 2, 26–50% positive cells, and 3, 51–100% positive cells. The scores for the intensity and the percentage of positive cells were multiplied to work out at a weighted score for each case. A score of 0–3 was estimated as low expression ( ), and scores of 4–9 indicated high expression (+). For b-catenin, the percentage of tumor cells with nucleus and cytoplasm staining was evaluated, and membrane staining did not enter the grading scheme.
Materials and methods Tissue samples Data of 80 patients with OSCC were included for current research. All the patients were pathologically diagnosed as oral squamous cell carcinoma, and had received radical resection of tumor and neck lymph node dissection at Tianjin Medical University Cancer Institute & Hospital between January 2006 and June 2008. In addition, 13 paraneoplastic tissues were used as control. Of the 80 OSCC patients, 52 were males (65.0%) and 28 were females (35.0%). The age ranged from 36 to 77 years, with a mean age of 58.7. clinico-pathological variables such as gender, age, tumor site, pathologic grading and clinical T staging, and lymph
Cell culture and transfection Tscca and Tca8113P160 (Human tongue squamous cell carcinoma cells) were purchased from the Chinese Academy of Medical Sciences Institute of Basic Medical Sciences and were grown in
Table 1 Expression of VHL and EMT related proteins were determined by immunohistochemistry, and clinico–pathologic variables in 80 OSCC. Variables
VHL Total
+
Gender Male Female
52 28
16 9
Age (y) 660 >60
54 26
Snail
N-cadherin
E-cadherin
MMP-2
P
+
P
+
P
+
17 13
0.226
35 14
17 14
0.130
23 9
29 19
0.293
31 18
35 15
19 11
0.538
36 13
18 13
0.152
24 8
30 18
0.242
0.843
17 15 11 6 1
10 11 4 4 1
0.883
15 17 11 4 2
12 9 4 6 0
0.377
13 7 6 4 2
14 19 9 6 0
17 12
0.878
26 24
20 10
0.702
25 24
21 10
0.140
18 14
17 29
22 12
0.183
23 27
16 14
0.525
20 28
19 13
0.121
16 35
19 10
0.003
18 32
17 13
0.071
14 35
21 10
0.001
b-Catenin P
+
P
+
36 19
0.899
31 20
21 8
0.294
35 15
17 8
37 18
0.949
33 18
21 8
0.479
Tumor localization Tongue 27 Gingiva 26 Oral floor 15 Check mucosa 10 Hard palate 2
10 9 3 1 2
17 17 12 9 0
0.116
17 18 8 7 1
10 8 7 3 1
T stage T1&T2 T3&T4
46 34
15 10
31 24
0.760
29 22
Pathology grade Grade 1 39 Grade 2&3 41
17 8
22 33
0.020
Lymphnode metastasis No 35 Yes 45
16 9
19 36
0.014
HIF-1a
MMP-9 P
+
P
+
21 10
0.683
34 16
31 18
23 8
0.309
0.246
14 15 12 7 1
13 11 3 3 1
28 20
0.853
24 25
17 13
22 28
0.273
21 11
14 34
0.001
P
18 12
0.468
39 20
13 8
0.729
35 15
19 11
0.538
40 19
14 7
0.924
0.403
17 15 11 6 1
10 11 4 4 1
0.874
23 17 10 9 0
4 9 5 1 2
0.052
22 9
0.053
27 23
19 11
0.414
29 30
17 4
0.011
23 26
16 15
0.684
21 29
18 12
0.119
26 33
13 8
0.160
16 33
19 12
0.012
16 34
19 11
0.006
22 37
13 8
0.051
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MEM and RPMI-1640 (Thermo Scientific, MA, USA) supplemented with 10% fetal bovine serum (FBS, Thermo Scientific), respectively. Cells were incubated in a humidified atmosphere of 5% CO2 and 95% air at 37 °C. HA-tagged wild-type VHL in pCDNA3 was kindly donated by Dr. Chunsheng Kang (Laboratory of Neuro-Oncology, Tianjin Neurological Institute). A pCDNA3 empty plasmid was used as a negative control. A total of 300 ng/ml plasmid was transfected with Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions.
Immunofluorescence (IF) Cells were seeded on cover-slips and fixed with 4% paraformaldehyde (PFA, Sigma, St. Louis, MO, USA), treated with 1% bovine serum albumin for 10 min and incubated with the b-catenin (Abmart) antibodies overnight at 4 °C. Fluorescein isothiocyanate-labeled secondary antibody (Zhongshan Bio Corp.) was added at 37 °C for 2 h. Diamidino-phenyl-indole reagent was used to stain the cell nuclei, and then the cells was visualized using FV-1000 laser scanning confocal microscopes (Olympus, Tokyo, Japan).
Western blotting After the cells were transfected and cultured for 48 h, they were thrice washed with phosphate buffer solution (PBS), and then were lysed in RIPA buffer (Millipore, MA, USA) or Nuclear and Cytoplasmic Extraction Reagents kit (Thermo Scientific) according to the manufacturer’s instructions. The protein concentrations of the supernatants were determined using the bicinchoninic acid protein assay. Typically, a 30 lg of protein was then separated by SDS-polyacrylamide gel electrophoresis. The gel was transferred to the polyvinylidene difluoride membrane with 250 mA at 4 °C for 1.5 h, using a wet electroblotting system (Bio-Rad, Hercules, CA, USA). The membranes were blocked in blocking buffer (5% nonfat dry milk, 0.1% Tween in PBS) for 1 h and then were incubated with primary antibodies and horseradish peroxidase-conjugated secondary antibodies (Zhongshan Bio Corp.). Immunoreactive protein bands were visualized using an enhanced chemi-luminescence detection system (SuperSignal ECL Kit, Thermo Scientific) and subsequent exposure of the membrane to Hyperfilm (Thermo Scientific). The same membrane was probed with mouse anti-b-actin or Histone H3 (Abmart) as a loading control. The densitometry of protein bands was measured using Image J software.
Statistical analysis Data were analyzed using SPSS 17.0. The interrelationship of VHL and EMT related proteins expression with clinico–pathologic variables was analyzed using v2 or Fisher exact test. Spearman’s correlation test was used to analyze the correlation of VHL with the EMT related proteins. Kaplan–Meier and time series test (log-rank test) were used for univariate survival analysis. Cox proportional hazards model was used for multivariate analysis. The measurement data were expressed as means ± s.e. Statistics was determined using the analysis of variance test. For all tests, differences were considered as significant at the value of P < 0.05.
MTT assay Tscca and Tca8113P160 cells were seeded in 96-well plates at a density of 4000 cells per well and were incubated with a 20 ll of methyl-thiazolyl tetrazolium (MTT) solution (5 mg/ml) before the transfection and 24, 48, 72 h after the transfection at 37 °C for 4 h. At the end of the incubation period, the solution was aspirated, and 150 ll DMSO were added to each well. The absorbance was measured at a wave length of 570 nm. Mean was determined for 8 replicate wells. Cell proliferation was calculated by absorbance of the transfection group/control group. Scratch assay Cells were cultured at the 6-well plates and incubated overnight. The scratches were created by scratching a straight line with a 20-ll tip vertically in the center of the dish. Dishes were washed with PBS once to remove the detached cells, and then the first image of the scratch was taken under microscope. Cells were treated as described. 24 and 48 h after the cell treatment, the width of the scratches was observed and measured using Image J software. The relative distance was calculated as a mean width of the cell scratch after transfection/the width before transfection. Transwell invasion assay Cell invasion assay was performed with BD invasion chambers (BD Bio, CA, USA). Briefly, a 50 ll of matrigel (BD Bio) and 200 ll of cell suspension (5 104/ml) was added into the inserts respectively. Medium containing 10% FBS was added to the lower chamber. The cells were cultured in a CO2 incubator at 37 °C for 48 h. The noninvasive cells in the upper chamber were wiped up with cotton swabs and invasive cells were fixed with 95% ethanol for 10 min and were stained with 0.5% crystal violet. Cells penetrated through the polyethylene terephthalate membrane were counted in ten representative microscopic fields (400 magnification).
Results Expression of VHL and EMT related proteins in the tissue samples of OSCC and the correlation of VHL and EMT related proteins with clinico-pathological features and prognosis of the patients Firstly, IHC assay was performed to assess the expression of VHL in 80 OSCC tissues and 13 para-neoplastic tissues. VHL protein had a high expression in 9 of all paraneoplastic tissues (69.2%), which was significantly higher compared to that in the OSCC tissues (25, 31.3%; P = 0.013). Then, the expression of EMT related proteins, and the VHL target protein, HIF-1a, were examined. The clinicopathological correlations with the expression of the abovementioned proteins were described in Table 1. The expression of VHL, b-catenin, N-cadherin, E-cadherin and MMP-2/9 were significantly associated with the lymph node metastasis (1A). Differences in VHL were significantly between the pathologic grades. Besides, an increase was noted in HIF-1a, from T1 & T2 stage to T3 & T4 stage. VHL was negatively correlated with b-catenin, N-cadherin, Snail, MMP-2/9, and HIF-1a, see Table 2. The Kaplan–Meier survival analysis showed that the pathologic grade, nodal status, and the expression of VHL, b-catenin, HIF-1a, MMP2 and MMP-9, were significantly associated with the survival time in the OSCC patients. Cox multivariate analysis indicated that low VHL expression, nodal status, and moderate and poor differentiation of the tumors were negatively correlated with the postoperative survival of these patients, suggesting that VHL is an independent risk factor and has impact on prognosis (Table 3, Fig. 1B).
Table 2 Spearman’s correlation test for correlation of VHL and EMT related proteins. r b-Catenin Snail N-cadherin E-cadherin MMP-2 MMP-9 HIF-1a
P 0.221 0.258 0.293 0.165 0.183 0.313 0.272
0.049 0.021 0.033 0.143 0.104 0.005 0.015
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Table 3 Univariate and multivariate analyses of prognostic factors in OSCC. Independent factors
Univariate (P)
Gender (Male/Female) Age (660/>60) Tumor localization T stage (T1&T2/T3&T4) Pathology grade (Grade 1/2&3) Lymph node metastasis (No/Yes) VHL ( /+) b-Catenin ( /+) Snail ( /+) N-cadherin ( /+) E-cadherin ( /+) MMP-2 ( /+) MMP-9 ( /+) HIF-1a ( /+)
0.166 0.222 0.208 0.074 0.000 0.000 0.002 0.041 0.399 0.092 0.223 0.033 0.037 0.025
Multivariate (P)
Relative risk
95%CI
0.001 0.038 0.019 0.629
2.978 1.977 0.443
1.599–5.547 1.038–3.766 0.225–0.874
0.527 0.721 0.158
Fig. 1. VHL and EMT related proteins expression in the groups with lymph node metastasis (LN+) and non-lymph node metastasis (LN ) and the correlation of VHL with patients’ overall survival. (A) IHC showed VHL positive staining was found in cytoplasm and nuclear, LN ( ) group showed stronger expression. b-Catenin was increased in nucleus and cytoplasm in LN (+). Snail and HIF-1a showed stronger nucleus and cytoplasm expression in LN (+) group. N-cadherin positive staining was obvious in LN (+) group, located in and membrane and cytoplasm. E-cadherin was located in membrane and expressed stronger in LN ( ). MMP-2/9 were showed stronger staining in cytoplasm in LN (+) (400). (B) Survival curves for patients with OSCC, in relation to VHL expressions. The patients with VHL high expression had a better prognosis. P < 0.05.
VHL re-expression results in inhibition of OSCC proliferation, migration and invasion in vitro To further investigate the biological role of VHL in modulating the OSCC growth in vitro, the Tscca and Tca8113P160 cell lines
were used in the study. Tscca and Tca8113P160 showed a low VHL expression. Thus, we elevated the VHL expression by transfecting a pCDNA3 plasmid carrying a VHL gene. 48 h after the transfection, the Western blot assay indicated the VHL expression was dramatically up-regulated comparing with the control and
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empty plasmid groups in both of the cell lines (Tscca: F = 193.927, P = 0.000; Tca8113P160: F = 88.724, P = 0.000, see Fig. 2A). The cells in the control and empty plasmid groups displayed an elongated shape and were separated from one another. Conversely, the morphology of transfected cells became flattened and grew in clusters and contacted more tightly (Fig. 2B). To further verify the role of VHL, MTT assay was used and demonstrated the VHL expression dramatically suppressed the cells proliferation of OSCC. The effect of growth-inhibition on VHL began at 24 h (Tscca: F = 64.685, P = 0.000; Tca8113P160: F = 110.937, P = 0.000), and reached the maximum 48 h after the transfection, as shown in Fig. 2C. The lowest survival rate was 69.4 ± 1.4% for Tscca cells (F = 924.237, P = 0.000) and 65.3 ± 1.3% for Tca8113P160 cells (F = 910.574, P = 0.000). Then, the scratch assay and transwell assay were performed to determine the motility and invasion in these cells. The relative distance was greater in pCDNA3-VHL groups than in the control and empty groups 24 h after the transfection (Tscca: F = 24.711, P = 0.001; Tca8113P160: F = 133.901, P = 0.000) and 48 h after the transfection (Tscca: F = 14.272, P = 0.005; Tca8113P160: F = 178.039, P = 0.000, Fig. 2D). Furthermore, a transwell assay showed that the number of invasion cells
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was 64.4 ± 4.27 and 61.3 ± 4.29 in the Tscca control group and empty group respectively, which was significantly higher than that in the pCDNA3-VHL group (42.0 ± 4.85, F = 120.437, P = 0.000). The number of the Tca8113P160 control and empty plasmid groups was 77.5 ± 3.66 and 74.3 ± 5.03 respectively, significantly higher compared to the pCDNA3-VHL group (51.6 ± 3.30, F = 73.316, P = 0.000, Fig. 2E). These results showed that the mesenchymal phenotype, proliferation, migration and invasion of the two cells was inhibited remarkably by the transfection of VHL plasmid, thus supported the notion that VHL is involved in the regulation of EMT process in OSCC. Restored expression of VHL suppresses EMT by interfering b-catenin activity IF staining showed a strong b-catenin signal in nucleus in control group cells. While in the recovered cells of VHL, nucleus b-catenin accumulation was significantly inhibited (Fig. 3A). The result was confirmed by Western blot assay for b-catenin expression in the nucleus. In cytoplasm, b-catenin decreased to some degree (Fig. 3B). To further explore the VHL’s role in EMT, the
Fig. 2. Restored expression of VHL changes cell morphological characteristics and inhibits cell proliferation, invasion and migration. (A) The morphology of cells changed after re-expression of VHL. (B) The expression of VHL was restored after transfection with VHL plasmid. (C) MTT assay. Restored VHL expression in Tscca and Tca8113P160 inhibited cell proliferation. (D) and (E) VHL expression suppressed cell migration and invasion. *P < 0.05.
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Fig. 2 (continued)
E-cadherin and N-cadherin expression in the pCDNA3-VHL-treated OSCC cells were examined. Importantly, in both the cell lines, the E-cadherin expression was elevated while the N-cadherin expression was obviously attenuated. In addition, Snail and MMP-2/9 were inhibited in the pCDNA3-VHL groups. These data suggested that the restored VHL expression might interrupt the EMT process in OSCC. Moreover, the Western blot analysis indicated that HIF-1a and VEGF were down-regulated accompanied with elevated expression of VHL (Fig. 3C). These results might explain the inhibitory action of the invasion and metastasis of tumors.
Discussion Quite a few previous studies have illustrated VHL acts as a tumor suppressor in human epithelial cancers, such as renal carcinoma, lung cancer, gallbladder carcinoma and glioblastoma.
Ectopic expression of VHL is believed to have a close relation with the tumorigenesis, progression, and prognosis of tumors [17–20]. However, the role of VHL in OSCC development remains unclear. In our study, it was discovered that the expression of VHL is lower in the OSCC samples than in the para-neoplastic tissues. The VHL expression predominates in the cases with well-differentiated lesions, without nodal metastasis. Besides, VHL expression is negatively correlated with the EMT related proteins. Moreover, the expression of EMT related proteins has a close relation with lymph node metastasis. However, our data showed that HIF-1a is more closely related to T stage than to the nodal status. These suggested that the low VHL expression has an effect on the OSCC metastasis via the EMT process. Univariate and multivariate survival analysis revealed that the moderate and poor differentiation of tumors, nodal metastasis and low expression of VHL may be the markers of OSCC with poor prognosis. Loss of VHL could induce the EMT process and might ultimately affect the prognosis of the patients.
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Fig. 3. Restored expression of VHL affected relative protein expression. (A) IF detection of b-catenin after transfection with VHL. (B) b-Catenin were decreased in both the cytoplasm and the nucleus. (C) Detection of relative protein in Tscca and Tca8113P160 cell after transfection with VHL by Western blot. b-Catenin, Snail, N-cadherin, HIF-1a, VEGF and MMP2/9 were down-regulated, the level of E-cadherin was upregulated. *P < 0.05.
To better understand the biological role of VHL, a gain function method was subsequently used by transfecting the VHL expression plasmid to Tscca and Tca8113P160. Chen et al. [21] transfected the wild-type VHL gene into the A498 and UMRC6 renal carcinoma cell lines, that lacked normal expression of the gene. The expression of VHL resulted in an overt suppression of the cell growth in the two
renal carcinoma cells, which for the first time provided a direct evidence that the VHL gene product could suppress the growth of renal carcinoma cells. Up to now, accumulating experimental evidence suggested that VHL may suppress the progression of tumor in many ways. Staller et al. [22] proved that the pVHL-negative A498(neo) cells can express a high level of chemokine receptor
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Fig. 3 (continued)
CXCR4 and can show a strong tendency to metastasize to specific secondary sites, whereas their VHL-expressing counterparts, A498(HA-pVHL30), do not Knockdown of pVHL by shRNA promotes EMT phenotype in A549 (Human lung adenocarcinoma cells) [18]. Based on the studies of breast cancers, degradation of VHL protein brings about the tumor angiogenesis and tumorigenesis [23]. In accordance with the findings, our results demonstrated that VHL provided a critical inhibition role in OSCC progression. The cells with re-expression of VHL endowed with a weaker capacity of proliferation, migration and invasion. Cells undergoing EMT acquire the mesenchymal phenotype and gain an increased motility and invasiveness. It has been shown in our findings that the VHL re-expression facilitates cells to reverse the mesenchymal characteristic. Concerning the relationship of VHL expression with the EMT related proteins and clinical outcome, it can be speculated that VHL could regulate EMT process in OSCC. Consistent with this hypothesis, we found that after transfection pCDNA3-VHL, the b-catenin expression was decreased in both the nucleus and the cytoplasm, especially in the nucleus. The b-catenin relocalization is one of the major oncogenic events and has been extensively studied in various human cancers, such as colorectal, breast and thyroid cancer [24]. Kim [25] used normal human corneal epithelial cells (HCE), MDCK, and DLD1 colon carcinoma cells to prove that Tcf/Lef could directly induce EMT when its transcription activity is activated by stable nuclear b-catenin. Studies of colorectal carcinomas have
shown that Wnt pathway stimulation triggers the translocation of b-catenin to the nucleus, where it can activate specific target genes, including genes involved in EMT, thus regulate the tumor proliferation, invasion and metastasis [26]. It has been demonstrated that pVHL is a component of an E3 complex that targets b-catenin for ubiquitination [27]. Peruzzi et al. [16] found that VHL can negatively regulated b-catenin in RCC cells, and VHL loss in clear cell RCC can promote oncogenic b-catenin signaling downstream of HGF. Resent study revealed that the Jade-1,which can be stabilized by pVHL, has been shown to be a novel E3 ubiquitin ligase that ubiquitinates b-catenin leading to its degradation. Proteasome inhibition completely abrogated the effect of Jade-1 on b-catenin, provided compelling evidence that VHL play an important role in b-catenin degradation via ubiquitination [28,29]. Moreover, It was found in our study that VHL re-expression could reduce the level of EMT markers such as Snai, N-cadherin, MMP2/9, could increase the level of E-cadherin. It confirmed the role of VHL in the inhibition of EMT by regulating b-catenin. In addition, an important function of VHL is to target cellular responses to oxygen, through its role in the oxygendependent inactivation of the transcription factor HIF, in particular HIF-1a [30,31]. Our results confirmed that the expression of HIF-1a and its downstream target, VEGF, were reduced in cells which VHL was restored. Recent reports pointed to HIF-1a as a regulator that integrates EMT [32], however, the interplay between b-catenin and HIF-1a may require further study.
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In conclusion, this is a preliminary study to explore the VHL’s role in EMT process of OSCC. The results mentioned above suggested that VHL is likely to regulate b-catenin, leading to OSCC’s EMT process. It could enhance the invasion and metastasis of OSCC, thus affect the patients’ prognosis. VHL may be a new marker for the progression and prognosis of OSCC, and it may provide a therapeutic target for clinical practice. Conflict of interest statement None declared. Acknowledgments This work was supported by a Grant from the National Natural Science Foundation of China (81172573, 81101916 and 51103107) and funds from Tianjin Science and Technology Committee (11JCYBJC10800 and 12JCYBJC33800). References [1] Warnakulasuriya S. Global epidemiology of oral and oropharyngeal cancer. Oral Oncol 2009;45(4–5):309–16. [2] Noguti J, De Moura CF, De Jesus GP, Da Silva VH, Hossaka TA, Oshima CT, et al. Metastasis from oral cancer: an overview. Cancer Genomics Proteomics 2012;9(5):329–35. [3] Olasz L, Szalma J, Orsi E, Tornoczky T, Marko T, Nyarady Z. Neoadjuvant chemotherapy: does it have benefits for the surgeon in the treatment of advanced squamous cell cancer of the oral cavity? Pathol Oncol Res: POR 2010;16(2):207–12. [4] Zhong LP, Zhang CP, Ren GX, Guo W, William Jr WN, Sun J, et al. Randomized phase III trial of induction chemotherapy with docetaxel, cisplatin, and fluorouracil followed by surgery versus up-front surgery in locally advanced resectable oral squamous cell carcinoma. J Clin Oncol: Off J Am Soc Clin Oncol 2013;31(6):744–51. [5] Smith A, Teknos TN, Pan Q. Epithelial to mesenchymal transition in head and neck squamous cell carcinoma. Oral Oncol 2013;49(4):287–92. [6] Chaw SY, Majeed AA, Dalley AJ, Chan A, Stein S, Farah CS. Epithelial to mesenchymal transition (EMT) biomarkers–E-cadherin, beta-catenin, APC and vimentin–in oral squamous cell carcinogenesis and transformation. Oral Oncol 2012;48(10):997–1006. [7] Wang B, Zhang CP, Zhang S, Yue K, Wang XD. Epithelial–mesenchymal transformation-mediated lymph node metastasis of oral squamous cell carcinoma and its mechanism. Chin J Clin Oncol 2012;39(23):1877–80. [8] Acloque H, Adams MS, Fishwick K, Bronner-Fraser M, Nieto MA. Epithelial– mesenchymal transitions: the importance of changing cell state in development and disease. J Clin Investig 2009;119(6):1438–49. [9] Zhao D, Tang XF, Yang K, Liu JY, Ma XR. Over-expression of integrin-linked kinase correlates with aberrant expression of Snail, E-cadherin and N-cadherin in oral squamous cell carcinoma: implications in tumor progression and metastasis. Clin Exp Metastasis 2012;29(8):957–69. [10] Iwai S, Yonekawa A, Harada C, Hamada M, Katagiri W, Nakazawa M, et al. Involvement of the Wnt–beta-catenin pathway in invasion and migration of oral squamous carcinoma cells. Int J Oncol 2010;37(5):1095–103. [11] Ferrara N. Vascular endothelial growth factor: molecular and biological aspects. Curr Top Microbiol Immunol 1999;237:1–30.
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[12] Young AC, Craven RA, Cohen D, Taylor C, Booth C, Harnden P, et al. Analysis of VHL gene alterations and their relationship to clinical parameters in sporadic conventional renal cell carcinoma. Clin Cancer Res: Off J Am Assoc Cancer Res 2009;15(24):7582–92. [13] Kim JH, Jung CW, Cho YH, Lee J, Lee SH, Kim HY, et al. Somatic VHL alteration and its impact on prognosis in patients with clear cell renal cell carcinoma. Oncol Rep 2005;13(5):859–64. [14] Jaakkola P, Mole DR, Tian YM, Wilson MI, Gielbert J, Gaskell SJ, et al. Targeting of HIF-alpha to the von Hippel–Lindau ubiquitylation complex by O2-regulated prolyl hydroxylation. Science 2001;292(5516):468–72. [15] Asakawa T, Esumi M, Endo S, Kida A, Ikeda M. Tongue cancer patients have a high frequency of allelic loss at the von Hippel–Lindau gene and other loci on 3p. Cancer 2008;112(3):527–34. [16] Peruzzi B, Athauda G, Bottaro DP. The von Hippel–Lindau tumor suppressor gene product represses oncogenic beta-catenin signaling in renal carcinoma cells. Proc Natl Acad Sci USA 2006;103(39):14531–6. [17] Yang Z, Yang Z, Xiong L, Huang S, Liu J, Yang L, et al. Expression of VHL and HIF1alpha and their clinicopathologic significance in benign and malignant lesions of the gallbladder. Appl Immunohistochem Mol Morphol: AIMM /Off Publ Soc Appl Immunohistochem 2011;19(6):534–9. [18] Zhou Q, Chen T, Ibe JC, Raj JU, Zhou G. Knockdown of von Hippel–Lindau protein decreases lung cancer cell proliferation and colonization. FEBS Lett 2012;586(10):1510–5. [19] Zia MK, Rmali KA, Watkins G, Mansel RE, Jiang WG. The expression of the von Hippel–Lindau gene product and its impact on invasiveness of human breast cancer cells. Int J Mol Med 2007;20(4):605–11. [20] Chen L, Han L, Zhang K, Shi Z, Zhang J, Zhang A, et al. VHL regulates the effects of miR-23b on glioma survival and invasion via suppression of HIF-1alpha/ VEGF and beta-catenin/Tcf-4 signaling. Neuro-oncology 2012;14(8):1026–36. [21] Chen F, Kishida T, Duh FM, Renbaum P, Orcutt ML, Schmidt L, et al. Suppression of growth of renal carcinoma cells by the von Hippel–Lindau tumor suppressor gene. Cancer Res 1995;55(21):4804–7. [22] Staller P, Sulitkova J, Lisztwan J, Moch H, Oakeley EJ, Krek W. Chemokine receptor CXCR4 downregulated by von Hippel–Lindau tumour suppressor pVHL. Nature 2003;425(6955):307–11. [23] Chou MT, Anthony J, Bjorge JD, Fujita DJ. The von Hippel–Lindau tumor suppressor protein is destabilized by src: implications for tumor angiogenesis and progression. Genes Cancer 2010;1(3):225–38. [24] Giles RH, van Es JH, Clevers H. Caught up in a Wnt storm: Wnt signaling in cancer. Biochim Biophys Acta 2003;1653(1):1–24. [25] Kim K, Lu Z, Hay ED. Direct evidence for a role of beta-catenin/LEF-1 signaling pathway in induction of EMT. Cell Biol Int 2002;26(5):463–76. [26] Sanchez-Tillo E, de Barrios O, Siles L, Cuatrecasas M, Castells A, Postigo A. Betacatenin/TCF4 complex induces the epithelial-to-mesenchymal transition (EMT)-activator ZEB1 to regulate tumor invasiveness. Proc Natl Acad Sci USA 2011;108(48):19204–9. [27] Polakis P. More than one way to skin a catenin. Cell 2001;105(5):563–6. [28] Chitalia VC, Foy RL, Bachschmid MM, Zeng L, Panchenko MV, Zhou MI, et al. Jade-1 inhibits Wnt signalling by ubiquitylating beta-catenin and mediates Wnt pathway inhibition by pVHL. Nat Cell Biol 2008;10(10):1208–16. [29] Zeng L, Bai M, Mittal AK, El-Jouni W, Zhou J, Cohen DM, et al. Candidate tumor suppressor and pVHL partner Jade-1 binds and inhibits AKT in renal cell carcinoma. Cancer Res 2013;73(17):5371–80. [30] Arjumand W, Sultana S. Role of VHL gene mutation in human renal cell carcinoma. Tumour Biol: J Int Soc Oncodev Biol Med 2012;33(1):9–16. [31] Harten SK, Esteban MA, Maxwell PH. Identification of novel VHL regulated genes by transcriptomic analysis of RCC10 renal carcinoma cells. Adv Enzyme Regul 2009;49(1):43–52. [32] Barriga EH, Maxwell PH, Reyes AE, Mayor R. The hypoxia factor HIF-1alpha controls neural crest chemotaxis and epithelial to mesenchymal transition. J Cell Biol 2013;201(5):759–76.
Contents lists available at ScienceDirect
Oral Oncology journal homepage: www.elsevier.com/locate/oraloncology
Loss of VHL expression contributes to epithelial–mesenchymal transition in oral squamous cell carcinoma Shu Zhang a,b,1, Xuan Zhou a,b,1, Bo Wang a,b, Kailiang Zhang c, Su Liu a,b, Kai Yue a,b, Lun Zhang a,b, Xudong Wang a,b,⇑ a b c
Department of Maxillofacial and E.N.T Oncology, Tianjin Medical University Cancer Institute & Hospital, Tianjin 300060, China National Clinical Research Center for Cancer, Key Laboratory of Cancer Prevention and Therapy, Tianjin 300060, China Department of Neurosurgery, Tianjin Medical University General Hospital, Laboratory of Neuro-oncology, Tianjin Neurological Institute, Tianjin 300052, China
a r t i c l e
i n f o
Article history: Received 25 January 2014 Received in revised form 18 May 2014 Accepted 6 June 2014 Available online 2 July 2014 Keywords: VHL Oral squamous cell carcinoma Epithelial–mesenchymal transition b-Catenin
s u m m a r y Objective: Loss of Von Hippel–Lindau (VHL) gene expression has been implicated in the development of human cancers. However, its function in oral squamous cell carcinoma (OSCC) remains undefined. The aim of this study was to clarify the VHL expression in OSCC and to explore the underlying mechanisms of VHL in modulating the epithelial–mesenchymal transition (EMT) in OSCC. Materials and methods: The expression of VHL, HIF-1a and EMT related proteins in OSCC tissues were evaluated by immunohistochemistry. The correlation of VHL with clinico-pathological characteristics, prognosis and EMT related proteins were analyzed. The roles of VHL on the cell morphology, proliferation, migration, and invasion were determined by MTT, scratch and transwell invasion assay in Tscca and Tca8113P160 cells. The EMT related proteins were determined by Western blot and immunofluorescence (IF) methods. Results: Loss of VHL expression was closely associated with pathologic grading, lymph node metastasis, poor prognosis, and EMT in OSCC. After re-expression of VHL, there was a cell morphologic change and motivation, proliferation, invasion of the cells were inhibited. The expression of Snail, N-cadherin and MMP-2/9, HIF-1a and VEGF were down-regulated in both the cell lines after transfection with VHL plasmid, while E-cadherin was up-regulated. Moreover, the effect of VHL suppressing b-catenin accumulation in nucleus was proved by Western blot and IF. Conclusion: VHL was significantly correlated with EMT process of OSCC. b-Catenin was an important downstream gene of VHL besides HIF-1a, which could induce the EMT process in OSCC. Ó 2014 Elsevier Ltd. All rights reserved.
Introduction Oral squamous cell carcinoma (OSCC) is a common type of malignant tumor in head and neck, with approximately 275,000 new cases worldwide each year [1]. It is characterized by a high degree of local invasiveness and a high rate of metastasis to the cervical lymph nodes [2]. Despite combined therapies, such as radical surgery, radiotherapy and neo-adjuvant chemotherapy, were used, the poor prognosis has not been significantly improved during the last decade. The 5-year overall survival rate is below 50% and is even lower in locally advanced or metastatic diseases ⇑ Corresponding author at: Department of Maxillofacial and E.N.T Oncology, Tianjin Medical University Cancer Institute & Hospital, Tianjin 300060, China. Tel./fax: +86 22 23340123. E-mail address: [email protected] (X. Wang). 1 These authors made equal contribution to this work. http://dx.doi.org/10.1016/j.oraloncology.2014.06.007 1368-8375/Ó 2014 Elsevier Ltd. All rights reserved.
[3,4]. Thus, it is urgent to promote the studies of the carcinogenesis and mechanism of metastasis in OSCC. It has been shown in our and others’ past researches that the process of epithelial–mesenchymal transition (EMT) is a key step for OSCC progression and metastasis [5–7]. Tumor cells undergoing EMT remodel from the epithelial phenotype to mesenchymal phenotype, accompanied by decrease in E-cadherin and increase in Snail, N-cadherin, vimentin and Twist, etc. [8]. These representative changes would facilitate cell losing their adhesiveness, dissolving the extracellular matrix that restrains them and spreading to the surrounding tissue. Moreover, the aforementioned typical markers of EMT are predictive of the patients’ survival [9]. The Wnt/b-catenin is one of the major signaling pathways in EMT. It was shown in a recent study that aberrant accumulated b-catenin in the cytoplasm is translocated to the nucleus where it binds to the Tcf/Lef, leading to the up-regulation of target genes, thus induce EMT in OSCC [10].
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The Von Hippel–Lindau gene (VHL) is the causal gene for VHL disease, which is a hereditary neoplastic syndrome, characterized by susceptibility for hyper-vascularized clear-cell renal cell carcinoma (ccRCC) [11]. Aberration in function of VHL gene, either through mutation, loss of heterozygosity (LOH), or methylation of VHL gene drives tumor initiation and progression [12,13]. It has been commonly known that VHL encoding product, VHL protein, acts as the recognition subunit of a multi-protein complex with ubiquitin E3 ligase activity, which targets the hypoxia inducible factor (HIF) for polyubiquitination and subsequent proteasomal degradation. Inactivation of VHL leads to HIF stabilization and activation that plays an important role in tumorigenesis [14]. Although evidence of abnormality in VHL inducing OSCC is evolving [15], little has been uncovered regarding the connection between VHL and EMT in OSCC. Based on earlier evidence that VHL gene deletion can activate b-catenin [16], an important mediator of EMT, we tested whether b-catenin played mediating role in loss of VHL regulated OSCC EMT process. In the study, we determined the expression of VHL, its target protein, HIF-1a, and EMT related proteins in the tissue samples of OSCC using immunohistochemistry (IHC), and attempted to elucidate the relationship between VHL and EMT in OSCC. Thus, we provided a novel mechanism of b-catenin that was regulated by VHL which could induce EMT in OSCC.
node status, were obtained from the medical records, as shown in Table 1. Survival information of the 80 patients was obtained from the visits by letters or by calls. Informed consent was obtained from all subjects. Immunohistochemistry (IHC) For IHC staining, paraffin-embedded tissue slides (4 lm thick) were deparaffinized, rehydrated and incubated with primary antibodies overnight at 4 °C. The following antibodies were used: VHL, HIF-1a, VEGF (Santa Cruz, CA, USA), Snail, N-cadherin, E-cadherin, matrix metalloproteina-2/9(MMP-2/9,Abcam, Cambridge, MA, USA), and b-catenin (Abmart, Shanghai, China). Then, they were incubated with biotin-labeled secondary antibody (Maxin Bio Corp., Fujian, China) for 1 h at room temperature and incubated with diaminobenzidine (Zhongshan Bio Corp., Beijing, China), counterstained with hematoxylin. Slides were dehydrated with different concentrations of alcohol and soaked in xylene, and then mounted with neutral balsam and visualized using light microscope. Ten representative fields at 400 magnification per slide were observed. The results were assessed by measuring both the staining intensity and the number of positive cells. The intensity of the positive reaction was scored, i.e. 0 = negative, 1 = light staining, 2 = moderate staining, and 3 = intense staining. Additionally, staining was scored on a scale of 0–3 according to the percentage of the cells involved: i.e. scale 0 means 0–5% positive cells; 1, 6–25% positive cells; 2, 26–50% positive cells, and 3, 51–100% positive cells. The scores for the intensity and the percentage of positive cells were multiplied to work out at a weighted score for each case. A score of 0–3 was estimated as low expression ( ), and scores of 4–9 indicated high expression (+). For b-catenin, the percentage of tumor cells with nucleus and cytoplasm staining was evaluated, and membrane staining did not enter the grading scheme.
Materials and methods Tissue samples Data of 80 patients with OSCC were included for current research. All the patients were pathologically diagnosed as oral squamous cell carcinoma, and had received radical resection of tumor and neck lymph node dissection at Tianjin Medical University Cancer Institute & Hospital between January 2006 and June 2008. In addition, 13 paraneoplastic tissues were used as control. Of the 80 OSCC patients, 52 were males (65.0%) and 28 were females (35.0%). The age ranged from 36 to 77 years, with a mean age of 58.7. clinico-pathological variables such as gender, age, tumor site, pathologic grading and clinical T staging, and lymph
Cell culture and transfection Tscca and Tca8113P160 (Human tongue squamous cell carcinoma cells) were purchased from the Chinese Academy of Medical Sciences Institute of Basic Medical Sciences and were grown in
Table 1 Expression of VHL and EMT related proteins were determined by immunohistochemistry, and clinico–pathologic variables in 80 OSCC. Variables
VHL Total
+
Gender Male Female
52 28
16 9
Age (y) 660 >60
54 26
Snail
N-cadherin
E-cadherin
MMP-2
P
+
P
+
P
+
17 13
0.226
35 14
17 14
0.130
23 9
29 19
0.293
31 18
35 15
19 11
0.538
36 13
18 13
0.152
24 8
30 18
0.242
0.843
17 15 11 6 1
10 11 4 4 1
0.883
15 17 11 4 2
12 9 4 6 0
0.377
13 7 6 4 2
14 19 9 6 0
17 12
0.878
26 24
20 10
0.702
25 24
21 10
0.140
18 14
17 29
22 12
0.183
23 27
16 14
0.525
20 28
19 13
0.121
16 35
19 10
0.003
18 32
17 13
0.071
14 35
21 10
0.001
b-Catenin P
+
P
+
36 19
0.899
31 20
21 8
0.294
35 15
17 8
37 18
0.949
33 18
21 8
0.479
Tumor localization Tongue 27 Gingiva 26 Oral floor 15 Check mucosa 10 Hard palate 2
10 9 3 1 2
17 17 12 9 0
0.116
17 18 8 7 1
10 8 7 3 1
T stage T1&T2 T3&T4
46 34
15 10
31 24
0.760
29 22
Pathology grade Grade 1 39 Grade 2&3 41
17 8
22 33
0.020
Lymphnode metastasis No 35 Yes 45
16 9
19 36
0.014
HIF-1a
MMP-9 P
+
P
+
21 10
0.683
34 16
31 18
23 8
0.309
0.246
14 15 12 7 1
13 11 3 3 1
28 20
0.853
24 25
17 13
22 28
0.273
21 11
14 34
0.001
P
18 12
0.468
39 20
13 8
0.729
35 15
19 11
0.538
40 19
14 7
0.924
0.403
17 15 11 6 1
10 11 4 4 1
0.874
23 17 10 9 0
4 9 5 1 2
0.052
22 9
0.053
27 23
19 11
0.414
29 30
17 4
0.011
23 26
16 15
0.684
21 29
18 12
0.119
26 33
13 8
0.160
16 33
19 12
0.012
16 34
19 11
0.006
22 37
13 8
0.051
811
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MEM and RPMI-1640 (Thermo Scientific, MA, USA) supplemented with 10% fetal bovine serum (FBS, Thermo Scientific), respectively. Cells were incubated in a humidified atmosphere of 5% CO2 and 95% air at 37 °C. HA-tagged wild-type VHL in pCDNA3 was kindly donated by Dr. Chunsheng Kang (Laboratory of Neuro-Oncology, Tianjin Neurological Institute). A pCDNA3 empty plasmid was used as a negative control. A total of 300 ng/ml plasmid was transfected with Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions.
Immunofluorescence (IF) Cells were seeded on cover-slips and fixed with 4% paraformaldehyde (PFA, Sigma, St. Louis, MO, USA), treated with 1% bovine serum albumin for 10 min and incubated with the b-catenin (Abmart) antibodies overnight at 4 °C. Fluorescein isothiocyanate-labeled secondary antibody (Zhongshan Bio Corp.) was added at 37 °C for 2 h. Diamidino-phenyl-indole reagent was used to stain the cell nuclei, and then the cells was visualized using FV-1000 laser scanning confocal microscopes (Olympus, Tokyo, Japan).
Western blotting After the cells were transfected and cultured for 48 h, they were thrice washed with phosphate buffer solution (PBS), and then were lysed in RIPA buffer (Millipore, MA, USA) or Nuclear and Cytoplasmic Extraction Reagents kit (Thermo Scientific) according to the manufacturer’s instructions. The protein concentrations of the supernatants were determined using the bicinchoninic acid protein assay. Typically, a 30 lg of protein was then separated by SDS-polyacrylamide gel electrophoresis. The gel was transferred to the polyvinylidene difluoride membrane with 250 mA at 4 °C for 1.5 h, using a wet electroblotting system (Bio-Rad, Hercules, CA, USA). The membranes were blocked in blocking buffer (5% nonfat dry milk, 0.1% Tween in PBS) for 1 h and then were incubated with primary antibodies and horseradish peroxidase-conjugated secondary antibodies (Zhongshan Bio Corp.). Immunoreactive protein bands were visualized using an enhanced chemi-luminescence detection system (SuperSignal ECL Kit, Thermo Scientific) and subsequent exposure of the membrane to Hyperfilm (Thermo Scientific). The same membrane was probed with mouse anti-b-actin or Histone H3 (Abmart) as a loading control. The densitometry of protein bands was measured using Image J software.
Statistical analysis Data were analyzed using SPSS 17.0. The interrelationship of VHL and EMT related proteins expression with clinico–pathologic variables was analyzed using v2 or Fisher exact test. Spearman’s correlation test was used to analyze the correlation of VHL with the EMT related proteins. Kaplan–Meier and time series test (log-rank test) were used for univariate survival analysis. Cox proportional hazards model was used for multivariate analysis. The measurement data were expressed as means ± s.e. Statistics was determined using the analysis of variance test. For all tests, differences were considered as significant at the value of P < 0.05.
MTT assay Tscca and Tca8113P160 cells were seeded in 96-well plates at a density of 4000 cells per well and were incubated with a 20 ll of methyl-thiazolyl tetrazolium (MTT) solution (5 mg/ml) before the transfection and 24, 48, 72 h after the transfection at 37 °C for 4 h. At the end of the incubation period, the solution was aspirated, and 150 ll DMSO were added to each well. The absorbance was measured at a wave length of 570 nm. Mean was determined for 8 replicate wells. Cell proliferation was calculated by absorbance of the transfection group/control group. Scratch assay Cells were cultured at the 6-well plates and incubated overnight. The scratches were created by scratching a straight line with a 20-ll tip vertically in the center of the dish. Dishes were washed with PBS once to remove the detached cells, and then the first image of the scratch was taken under microscope. Cells were treated as described. 24 and 48 h after the cell treatment, the width of the scratches was observed and measured using Image J software. The relative distance was calculated as a mean width of the cell scratch after transfection/the width before transfection. Transwell invasion assay Cell invasion assay was performed with BD invasion chambers (BD Bio, CA, USA). Briefly, a 50 ll of matrigel (BD Bio) and 200 ll of cell suspension (5 104/ml) was added into the inserts respectively. Medium containing 10% FBS was added to the lower chamber. The cells were cultured in a CO2 incubator at 37 °C for 48 h. The noninvasive cells in the upper chamber were wiped up with cotton swabs and invasive cells were fixed with 95% ethanol for 10 min and were stained with 0.5% crystal violet. Cells penetrated through the polyethylene terephthalate membrane were counted in ten representative microscopic fields (400 magnification).
Results Expression of VHL and EMT related proteins in the tissue samples of OSCC and the correlation of VHL and EMT related proteins with clinico-pathological features and prognosis of the patients Firstly, IHC assay was performed to assess the expression of VHL in 80 OSCC tissues and 13 para-neoplastic tissues. VHL protein had a high expression in 9 of all paraneoplastic tissues (69.2%), which was significantly higher compared to that in the OSCC tissues (25, 31.3%; P = 0.013). Then, the expression of EMT related proteins, and the VHL target protein, HIF-1a, were examined. The clinicopathological correlations with the expression of the abovementioned proteins were described in Table 1. The expression of VHL, b-catenin, N-cadherin, E-cadherin and MMP-2/9 were significantly associated with the lymph node metastasis (1A). Differences in VHL were significantly between the pathologic grades. Besides, an increase was noted in HIF-1a, from T1 & T2 stage to T3 & T4 stage. VHL was negatively correlated with b-catenin, N-cadherin, Snail, MMP-2/9, and HIF-1a, see Table 2. The Kaplan–Meier survival analysis showed that the pathologic grade, nodal status, and the expression of VHL, b-catenin, HIF-1a, MMP2 and MMP-9, were significantly associated with the survival time in the OSCC patients. Cox multivariate analysis indicated that low VHL expression, nodal status, and moderate and poor differentiation of the tumors were negatively correlated with the postoperative survival of these patients, suggesting that VHL is an independent risk factor and has impact on prognosis (Table 3, Fig. 1B).
Table 2 Spearman’s correlation test for correlation of VHL and EMT related proteins. r b-Catenin Snail N-cadherin E-cadherin MMP-2 MMP-9 HIF-1a
P 0.221 0.258 0.293 0.165 0.183 0.313 0.272
0.049 0.021 0.033 0.143 0.104 0.005 0.015
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Table 3 Univariate and multivariate analyses of prognostic factors in OSCC. Independent factors
Univariate (P)
Gender (Male/Female) Age (660/>60) Tumor localization T stage (T1&T2/T3&T4) Pathology grade (Grade 1/2&3) Lymph node metastasis (No/Yes) VHL ( /+) b-Catenin ( /+) Snail ( /+) N-cadherin ( /+) E-cadherin ( /+) MMP-2 ( /+) MMP-9 ( /+) HIF-1a ( /+)
0.166 0.222 0.208 0.074 0.000 0.000 0.002 0.041 0.399 0.092 0.223 0.033 0.037 0.025
Multivariate (P)
Relative risk
95%CI
0.001 0.038 0.019 0.629
2.978 1.977 0.443
1.599–5.547 1.038–3.766 0.225–0.874
0.527 0.721 0.158
Fig. 1. VHL and EMT related proteins expression in the groups with lymph node metastasis (LN+) and non-lymph node metastasis (LN ) and the correlation of VHL with patients’ overall survival. (A) IHC showed VHL positive staining was found in cytoplasm and nuclear, LN ( ) group showed stronger expression. b-Catenin was increased in nucleus and cytoplasm in LN (+). Snail and HIF-1a showed stronger nucleus and cytoplasm expression in LN (+) group. N-cadherin positive staining was obvious in LN (+) group, located in and membrane and cytoplasm. E-cadherin was located in membrane and expressed stronger in LN ( ). MMP-2/9 were showed stronger staining in cytoplasm in LN (+) (400). (B) Survival curves for patients with OSCC, in relation to VHL expressions. The patients with VHL high expression had a better prognosis. P < 0.05.
VHL re-expression results in inhibition of OSCC proliferation, migration and invasion in vitro To further investigate the biological role of VHL in modulating the OSCC growth in vitro, the Tscca and Tca8113P160 cell lines
were used in the study. Tscca and Tca8113P160 showed a low VHL expression. Thus, we elevated the VHL expression by transfecting a pCDNA3 plasmid carrying a VHL gene. 48 h after the transfection, the Western blot assay indicated the VHL expression was dramatically up-regulated comparing with the control and
S. Zhang et al. / Oral Oncology 50 (2014) 809–817
empty plasmid groups in both of the cell lines (Tscca: F = 193.927, P = 0.000; Tca8113P160: F = 88.724, P = 0.000, see Fig. 2A). The cells in the control and empty plasmid groups displayed an elongated shape and were separated from one another. Conversely, the morphology of transfected cells became flattened and grew in clusters and contacted more tightly (Fig. 2B). To further verify the role of VHL, MTT assay was used and demonstrated the VHL expression dramatically suppressed the cells proliferation of OSCC. The effect of growth-inhibition on VHL began at 24 h (Tscca: F = 64.685, P = 0.000; Tca8113P160: F = 110.937, P = 0.000), and reached the maximum 48 h after the transfection, as shown in Fig. 2C. The lowest survival rate was 69.4 ± 1.4% for Tscca cells (F = 924.237, P = 0.000) and 65.3 ± 1.3% for Tca8113P160 cells (F = 910.574, P = 0.000). Then, the scratch assay and transwell assay were performed to determine the motility and invasion in these cells. The relative distance was greater in pCDNA3-VHL groups than in the control and empty groups 24 h after the transfection (Tscca: F = 24.711, P = 0.001; Tca8113P160: F = 133.901, P = 0.000) and 48 h after the transfection (Tscca: F = 14.272, P = 0.005; Tca8113P160: F = 178.039, P = 0.000, Fig. 2D). Furthermore, a transwell assay showed that the number of invasion cells
813
was 64.4 ± 4.27 and 61.3 ± 4.29 in the Tscca control group and empty group respectively, which was significantly higher than that in the pCDNA3-VHL group (42.0 ± 4.85, F = 120.437, P = 0.000). The number of the Tca8113P160 control and empty plasmid groups was 77.5 ± 3.66 and 74.3 ± 5.03 respectively, significantly higher compared to the pCDNA3-VHL group (51.6 ± 3.30, F = 73.316, P = 0.000, Fig. 2E). These results showed that the mesenchymal phenotype, proliferation, migration and invasion of the two cells was inhibited remarkably by the transfection of VHL plasmid, thus supported the notion that VHL is involved in the regulation of EMT process in OSCC. Restored expression of VHL suppresses EMT by interfering b-catenin activity IF staining showed a strong b-catenin signal in nucleus in control group cells. While in the recovered cells of VHL, nucleus b-catenin accumulation was significantly inhibited (Fig. 3A). The result was confirmed by Western blot assay for b-catenin expression in the nucleus. In cytoplasm, b-catenin decreased to some degree (Fig. 3B). To further explore the VHL’s role in EMT, the
Fig. 2. Restored expression of VHL changes cell morphological characteristics and inhibits cell proliferation, invasion and migration. (A) The morphology of cells changed after re-expression of VHL. (B) The expression of VHL was restored after transfection with VHL plasmid. (C) MTT assay. Restored VHL expression in Tscca and Tca8113P160 inhibited cell proliferation. (D) and (E) VHL expression suppressed cell migration and invasion. *P < 0.05.
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Fig. 2 (continued)
E-cadherin and N-cadherin expression in the pCDNA3-VHL-treated OSCC cells were examined. Importantly, in both the cell lines, the E-cadherin expression was elevated while the N-cadherin expression was obviously attenuated. In addition, Snail and MMP-2/9 were inhibited in the pCDNA3-VHL groups. These data suggested that the restored VHL expression might interrupt the EMT process in OSCC. Moreover, the Western blot analysis indicated that HIF-1a and VEGF were down-regulated accompanied with elevated expression of VHL (Fig. 3C). These results might explain the inhibitory action of the invasion and metastasis of tumors.
Discussion Quite a few previous studies have illustrated VHL acts as a tumor suppressor in human epithelial cancers, such as renal carcinoma, lung cancer, gallbladder carcinoma and glioblastoma.
Ectopic expression of VHL is believed to have a close relation with the tumorigenesis, progression, and prognosis of tumors [17–20]. However, the role of VHL in OSCC development remains unclear. In our study, it was discovered that the expression of VHL is lower in the OSCC samples than in the para-neoplastic tissues. The VHL expression predominates in the cases with well-differentiated lesions, without nodal metastasis. Besides, VHL expression is negatively correlated with the EMT related proteins. Moreover, the expression of EMT related proteins has a close relation with lymph node metastasis. However, our data showed that HIF-1a is more closely related to T stage than to the nodal status. These suggested that the low VHL expression has an effect on the OSCC metastasis via the EMT process. Univariate and multivariate survival analysis revealed that the moderate and poor differentiation of tumors, nodal metastasis and low expression of VHL may be the markers of OSCC with poor prognosis. Loss of VHL could induce the EMT process and might ultimately affect the prognosis of the patients.
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Fig. 3. Restored expression of VHL affected relative protein expression. (A) IF detection of b-catenin after transfection with VHL. (B) b-Catenin were decreased in both the cytoplasm and the nucleus. (C) Detection of relative protein in Tscca and Tca8113P160 cell after transfection with VHL by Western blot. b-Catenin, Snail, N-cadherin, HIF-1a, VEGF and MMP2/9 were down-regulated, the level of E-cadherin was upregulated. *P < 0.05.
To better understand the biological role of VHL, a gain function method was subsequently used by transfecting the VHL expression plasmid to Tscca and Tca8113P160. Chen et al. [21] transfected the wild-type VHL gene into the A498 and UMRC6 renal carcinoma cell lines, that lacked normal expression of the gene. The expression of VHL resulted in an overt suppression of the cell growth in the two
renal carcinoma cells, which for the first time provided a direct evidence that the VHL gene product could suppress the growth of renal carcinoma cells. Up to now, accumulating experimental evidence suggested that VHL may suppress the progression of tumor in many ways. Staller et al. [22] proved that the pVHL-negative A498(neo) cells can express a high level of chemokine receptor
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Fig. 3 (continued)
CXCR4 and can show a strong tendency to metastasize to specific secondary sites, whereas their VHL-expressing counterparts, A498(HA-pVHL30), do not Knockdown of pVHL by shRNA promotes EMT phenotype in A549 (Human lung adenocarcinoma cells) [18]. Based on the studies of breast cancers, degradation of VHL protein brings about the tumor angiogenesis and tumorigenesis [23]. In accordance with the findings, our results demonstrated that VHL provided a critical inhibition role in OSCC progression. The cells with re-expression of VHL endowed with a weaker capacity of proliferation, migration and invasion. Cells undergoing EMT acquire the mesenchymal phenotype and gain an increased motility and invasiveness. It has been shown in our findings that the VHL re-expression facilitates cells to reverse the mesenchymal characteristic. Concerning the relationship of VHL expression with the EMT related proteins and clinical outcome, it can be speculated that VHL could regulate EMT process in OSCC. Consistent with this hypothesis, we found that after transfection pCDNA3-VHL, the b-catenin expression was decreased in both the nucleus and the cytoplasm, especially in the nucleus. The b-catenin relocalization is one of the major oncogenic events and has been extensively studied in various human cancers, such as colorectal, breast and thyroid cancer [24]. Kim [25] used normal human corneal epithelial cells (HCE), MDCK, and DLD1 colon carcinoma cells to prove that Tcf/Lef could directly induce EMT when its transcription activity is activated by stable nuclear b-catenin. Studies of colorectal carcinomas have
shown that Wnt pathway stimulation triggers the translocation of b-catenin to the nucleus, where it can activate specific target genes, including genes involved in EMT, thus regulate the tumor proliferation, invasion and metastasis [26]. It has been demonstrated that pVHL is a component of an E3 complex that targets b-catenin for ubiquitination [27]. Peruzzi et al. [16] found that VHL can negatively regulated b-catenin in RCC cells, and VHL loss in clear cell RCC can promote oncogenic b-catenin signaling downstream of HGF. Resent study revealed that the Jade-1,which can be stabilized by pVHL, has been shown to be a novel E3 ubiquitin ligase that ubiquitinates b-catenin leading to its degradation. Proteasome inhibition completely abrogated the effect of Jade-1 on b-catenin, provided compelling evidence that VHL play an important role in b-catenin degradation via ubiquitination [28,29]. Moreover, It was found in our study that VHL re-expression could reduce the level of EMT markers such as Snai, N-cadherin, MMP2/9, could increase the level of E-cadherin. It confirmed the role of VHL in the inhibition of EMT by regulating b-catenin. In addition, an important function of VHL is to target cellular responses to oxygen, through its role in the oxygendependent inactivation of the transcription factor HIF, in particular HIF-1a [30,31]. Our results confirmed that the expression of HIF-1a and its downstream target, VEGF, were reduced in cells which VHL was restored. Recent reports pointed to HIF-1a as a regulator that integrates EMT [32], however, the interplay between b-catenin and HIF-1a may require further study.
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