|Year : 2016 | Volume
| Issue : 1 | Page : 20-24
P120ctn may participate in epithelial-mesenchymal transition in OSCC
C Zhong1, Z Zuo2, Qing Ji3, D Feng1
1 Chongqing Key Laboratory for Oral Diseases and Biomedical Sciences, Stomatology Hospital of Chongqing Medical University, Chongqing, China
2 State Key Laboratory of Cellular Stress Biology, School of Life Sciences, Xiamen University, Xiamen, China
3 Department of Stomatology, Xiamen Medical College, Xiamen, China
|Date of Web Publication||28-Apr-2016|
Chongqing Key Laboratory for Oral Diseases and Biomedical Sciences, Stomatology Hospital of Chongqing Medical University, Chongqing
Source of Support: None, Conflict of Interest: None
Aim: Oral squamous cell carcinoma (OSCC) is the eighth most common cause of cancer death. OSCC cells can easily invade tissues and metastasize to the cervical lymph nodes. Understanding the molecular basis of OSCC metastasis would facilitate the development of new therapeutic approaches to the disease. Materials and Methods: To identify the potential role of catenin (P120ctn) in the progression of OSCC, HN4 cells (derived from a primary OSCC) and HN12 cells (derived from a lymph node metastasis in the same patient) were used in the present study. The samples of 28 patients with OSCC were examined to determine the expression of P120ctn and E-cadherin (E-cad; E: Epithelial) in vivo. Then, P120ctn was subsequently knocked down in HN4 cells (HN4/shP120ctn) and overexpressed in HN12 cells (HN12/P120ctn). Invasion and migration capacity, as well as the expression of the epithelial-to-mesenchymal transition (EMT) markers N-cadherin and vimentin were detected. Results: The results showed a positive correlation between the expression of P120ctn and E-cad in OSCC samples. The overexpression of P120ctn led to a decrease in both invasion and migration capacity of HN12 cells accompanied by a decrease in EMT markers. In contrast, knockdown of P120ctn led to an increase in both invasion and migration capacity accompanied by an increase in EMT markers. Conclusion: Considered together, we concluded that P120ctn might regulate EMT in OSCC through E-cad. The proper expression of P120ctn might therefore serve as a therapeutic goal for the inhibition of OSCC progression.
Keywords: Epithelial-mesenchymal transition, invasion, migration, oral squamous cell carcinoma, P120ctn
|How to cite this article:|
Zhong C, Zuo Z, Ji Q, Feng D. P120ctn may participate in epithelial-mesenchymal transition in OSCC. Indian J Cancer 2016;53:20-4
| » Introduction|| |
Oral squamous cell carcinoma (OSCC) is the eighth most common cause of cancer death. OSCC cells can easily invade tissues and metastasize to the cervical lymph nodes. Epithelial–mesenchymal transition (EMT) is a normal biological process during developmental stages, but it is also involved in cancer invasion and metastasis. It is characterized by the loss of typical epithelial cell markers such as E-cadherin (E-cad; E: Epithelial) and the gain of mesenchymal markers such as vimentin. The induction of EMT in squamous carcinoma cells drives tumor progression by enhancing cell-invasive and metastatic features., Takkunen et al. report that the downregulation of E-cad correlates with the progression of EMT in OSCC cells. As the core molecule of adherens junctions, E-cad connects neighboring epithelial cells by calcium-dependent homotypic interactions through its extracellular tail. Loss of its expression causes detachment of cells, the loss of epithelial differentiation, and the acquisition of a mesenchymal phenotype, which contributes to metastasis and invasion.
P120 catenin (P120ctn), a member of the armadillo/β-catenin gene family, binds to the juxtamembrane domain (JMD) of E-cad with other catenins. The E-cad/catenin complex, with P120ctn and E-cad as key components, plays a crucial role in stabilizing cell adhesions and cytoskeletal remodeling during tumor growth., Recent evidence indicates that the complete loss, downregulation, or mislocalization of P120ctn regulates the expression level of E-cad and correlates with the progression of different types of human tumors. Although a strong correlation between the loss of membranous immunoreactivity of P120ctn and a high grade of dedifferentiation is observed in OSCC cell lines, a direct correlation between the abnormal expression of P120ctn with E-cad and EMT has not been shown in OSCC.
In this study, OSCC samples were immunochemically examined to determine the expression of P120ctn and E-cad in vivo. We overexpressed and silenced P120ctn expression in oral carcinoma cell lines to determine the relationship between P120ctn and E-cad and the effect they have on the EMT of OSCC.
| » Materials and Methods|| |
Cell lines and culture conditions
HN4 cells, originating from a primary OSCC of the tongue, and HN12 cells, derived from a lymph node metastasis in the same patient, were highly invasive in vitro and tumorigenic in vivo. The cells were obtained from Michigan State University and cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum, 100 IU/mL penicillin (Sigma, St. Louis, MO), and 100 μg/mL streptomycin (Sigma) at 37°C in 95% air/5% carbon dioxide (CO2). Human oral keratinocytes (HOK) were obtained from EIAab Science (Wuhan, China) and cultured in a similar environment.
HN12 cell lines were transfected with P120ctn complementary deoxyribonucleic acid (cDNA) (OriGene, Rockville, MD) and named HN12/P120ctn using Lipofectamine (Invitrogen, Carlsbad, CA). An empty plasmid was used as a negative control. HN4 cell lines were transfected with P120ctn-shRNA plasmid (OriGene, Rockville, MD) and named HN4/shP120ctn. A nonsilencing short hairpin ribonucleic acid (shRNA) plasmid was used as a negative control.
RNA extraction and reverse transcription
Total RNA was extracted from HOK, HN4, and HN12 cells using TRIzol (Invitrogen, Carlsbad, CA) according to the protocol of the manufacturer. First-strand cDNA was synthesized from 2 mg of the total RNA using a Revert Aid Mu-MLV cDNA synthesis kit (Toyobo, Japan) according to the protocol of the manufacturer.
Real-time quantitative PCR
Real-time quantitative polymerase chain reaction (qRT-PCR) analysis was performed on a Rotor-gene 6000 real-time PCR system (ABI, USA) using SYBR Green I fluorescent dye. The reactions were performed in a 10 μL volume mix containing 0.2 μL SYBR Green I, 5 pmol L −1 specific primers, and approximately 50 ng cDNA. The cycling parameters were 95°C for 10 minutes, followed by 45 cycles of 95°C for 20 seconds, 55°C for 20 seconds, and 72°C for 20 seconds. Threshold cycles and dissociation curves were determined with Rotor-gene 6000 software, to confirm that only one PCR product was amplified and detected, and gene expression levels were normalized to β-actin. Standard curves and primer efficiencies were determined for all genes analyzed by qRT-PCR. Primer sequences, designed using software Primer Premier 5.0, are shown in [Table 1].
Western blotting assay
Total protein, extracted from HN4, HN4/shP120ctn, HN12, and HN12/P120ctn cells (n = 3 per condition) were analyzed using anti-P120ctn antibodies from Abcam (Cambridge, MA) and anti-Ed-cad antibodies from (Santa Cruz Biotechnology, Santa Cruz, CA). The protein band, specifically bound to the primary antibody, was detected using an antirabbit (P120ctn) or antirat (E-cad) immunoglobulin G-alkaline phosphatase (IgG-AP)–linked antibody.
Immunohistochemical staining and evaluation
We obtained tumors from 28 patients with OSCC. These patients had undergone surgery in the Department of Oral and Maxillary Surgery at Zhongshan Hospital of Xiamen University between 1999 and 2005, and had not undergone any previous therapy. All samples were subjected to fixation in 100 ml/L formalin and cut into 4 μm serial sections for immunostaining performed by the streptavidin–peroxidase (SP) method. Anti-P120ctn antibodies (1:500 dilution, Abcam, Cambridge, MA) and anti-E-cad antibodies (1:500 dilution, Santa Cruz Biotechnology, Santa Cruz, CA) were used for incubations overnight at 4°C. Biotinylated secondary antibodies and diaminobenzidine (DAB) were purchased from Thermo Fisher Scientific (Waltham, MA, USA) and Boster Bioengineering (Wuhan, China), respectively.
Assessment of P120ctn and E-cad immunoreactivity
The expression of P120ctn and E-cad were evaluated by the percentage of positive cells (0-100%) for membranous and cytoplasmic staining separately. 'Normal expression' and 'reduced membranous expression' were defined as the tumor cell membrane staining over 90% and less than 90%, respectively. When more than 10% of the tumor cells were cytoplasmically stained, the sample was labeled as 'ectopic cytoplasmic expression'. A designation of either 'reduced membranous expression' or 'ectopic cytoplasmic expression' was used to define the abnormal expression of P120ctn and E-cad.
Cell migration and invasion assays
Cells (2.5 × 105) were plated on uncoated upper chambers of Transwell plates (Corning, NY) for migration assays. Cells (2.5 × 105) were placed on Matrigel (1:4 dilution; BD Biosciences, San Jose, CA)-coated upper chambers for invasion assays.
The medium in the top component was aspirated and replaced with serum-free media six hours after seeding, and the medium containing 20% serum was used as a chemoattractant in the lower chambers. After 24-hour incubation at 37°C in 5% CO2, cells that passed through the filter were stained with crystal violet (C3886, Sigma) and photographed before being counted in 10 random high-power fields. Each experiment was done three times independently.
All data were presented as the means ± standard deviation. The presence of statistical differences among groups was determined using analysis of variance (ANOVA), and the method of the least significant difference (Dunnett test) was used to compare each group and the control. A linear regression was carried out to verify the concentration effect. All of the statistical analyses were carried out using the statistical software SPSS 13.0 for Windows. Statistical significance was recognized when P < 0.05.
| » Results|| |
Abnormal expression of P120ctn and E-cad in OSCC tissues
We examined the expression of P120ctn and E-cad in oral squamous cancers. As shown in [Figure 1], P120ctn and E-cad showed strong and continuous immunohistochemical staining of the membrane in normal oral tissues. In contrast, we found decreased or absent membrane expression with increased cytoplasmic staining for P120ctn and E-cad in oral squamous cancers.
|Figure 1: P120 catenin immunoreactivity in normal oral tissues (a) and in oral squamous cell carcinoma (OSCC) (c); E-cadherin immunoreactivity in normal oral tissues (b) and in OSCC (d); scale bar: 100 um|
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As shown in [Table 2], the abnormal expression of P120ctn and E-cad was significantly higher in patients with lymph node metastasis than patients without lymph node metastasis (P < 0.05). The rate for abnormal expression of P120ctn and E-cad in patients with moderate cell differentiation was 84.62 and 92.31%, respectively. Patients with well-differentiated cells, in contrast, had abnormal expression rates of 40.00 and 26.67% for P120ctn and E-cad, respectively (P < 0.05). There were no significant correlations between the expression of P120ctn and E-cad with TNM stages of the tumor (all P > 0.05).
|Table 2: Correlations between expression of Pl20ctn and E-cad with clinicopathological parameters in OSCCs|
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As shown in [Table 3], the expression of E-cad significantly correlated with the expression of P120ctn (P < 0.05). There was a significant concordance between the expression of E-cad and P120ctn.
Expression of P120ctn and E-cad in OSCC cells
To further evaluate the protein expression of P120ctn and E-cad, we performed a western blotting assay using the total protein extracted from HOK, HN4, and HN12 cells. As shown in [Figure 2]a we observed a low expression of P120ctn and E-cad in HN4 cells compared with HOK cells (P < 0.05). We also found that the expression of P120ctn and E-cad was higher in HN4 than HN12 cells (P < 0.05).
|Figure 2: Levels of P120 catenin and E-cadherin protein (a) and messenger ribonucleic acid (b) by western blotting analysis and real-time quantitative polymerase chain reaction analysis in HOK, HN4, HN12 cells|
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Analysis of the expression of P120ctn and E-cad mRNA in HN4 and HN12 cells
The transcriptional levels of P120ctn and E-cad were determined with qRT-PCR assays in HOK, HN4, and HN12 cells. As shown in [Figure 2]b, consistent with western blotting results, P120ctn and E-cad were highly expressed in the HOK cell lines compared with HN4 and HN12 cells. In addition, we found that in the HN12 cell line, which exhibited a higher migration capacity than HN4, the abnormal expression of E-cad and P120ctn was more obvious.
Inhibition of the EMT of OSCC by P120ctn
To test the effect of P120ctn and E-cad on the EMT of OSCC, we determined the expression of P120ctn, E-cad, N-cadherin (mesenchymal cadherin), and vimentin (mesenchymal marker) in HN4, HN4/shP120ctn, HN12, and HN12/P120ctn cells by western blotting. As shown in [Figure 3], the expression of E-cad increased dramatically in HN12/P120ctn cells (compared with HN12 cells) and decreased in HN4/shP120ctn cells (compared with HN4 cells), indicating that the loss of E-cad protein expression correlates with the decrease in P120ctn expression. On the other hand, an inverse relationship between the expression of P120ctn and mesenchymal markers (N-cadherin and vimentin) was observed, suggesting that P120ctn inhibits the acquisition of a mesenchymal phenotype in OSCC.
|Figure 3: Western blotting analysis of P120 catenin (P120ctn), E-cadherin, N-cadherin, and vimentin in HN4, HN4/shP120ctn, HN12 and HN12/P120ctn cells|
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Inhibition of the invasion and migration of OSCC by P120ctn
To further investigate the role of P120ctn in OSCC tumor progression, we examined the ability of cells expressing different levels of P120ctn to migrate and invade in vitro. The cells were plated on uncoated Transwell filters and allowed to migrate for 24 hours. As shown in [Figure 4], the numbers of HN4, HN4/shP120ctn, HN12, and HN12/P120ctn cells in the lower chamber were 58.23 ± 13.67, 118.60 ± 18.63, 125.50 ± 19.88, and 56.63 ± 10.61, respectively. The HN4/shP120ctn cells showed an increased migration capacity compared with HN4 cells, and HN12/P120ctn cells showed a decreased migration capacity compared with HN12 cells. Similarly, as shown in [Figure 5], in the invasion assay, the numbers of HN4, HN4/shP120ctn, HN12, and HN12/P120ctn cells in the lower chamber were 51.63 ± 15.09, 102.34 ± 23.58, 121.67 ± 20.77, and 41.66 ± 9.28, respectively. The invasion and migration capacity of HN4 cells increased markedly after the inhibition of P120ctn expression. In contrast, the invasion and migration capacity of HN12 cells decreased markedly when P120ctn was overexpressed (all P < 0.05). These results show that P120ctn may play a key role in the inhibition of invasion and migration in oral squamous cell carcinoma.
|Figure 4: Migration capacity of HN4 cells was markedly increased after inhibition of the expression of P120 catenin (P120ctn), and that of HN12 was decreased after P120ctn was overexpressed (a, c); representative images of the cultures from migration assays (b, d); quantification of the migration assay; scale bar: 50 um|
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|Figure 5: Invasion capacity of HN4 cells was markedly increased after inhibition of the expression of P120 catenin (P120ctn), and that of HN12 was decreased after P120ctn was overexpressed (a, c); representative images of the cultures from invasion assays (b, d); quantification of the invasion assay; scale bar: 50 um|
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| » Discussion|| |
Abnormal expression of p120ctn and E-cad in tumor cells has been implicated in tumor progression; however, the relationship between the expression of these molecules, as well as their correlation with clinical and pathological factors of cancers, are poorly defined. Our results showed a positive correlation between the abnormal expression of P120ctn and E-cad in OSCC samples. The overexpression of P120ctn led to a decrease in both invasion and migration capacity of HN12 cells accompanied by a decrease in EMT markers. In contrast, knockdown of P120ctn led to an increase in both invasion and migration capacity accompanied by an increase in EMT markers.
We found that the abnormal expression rate of P120ctn was significantly higher in OSCC than the normal oral mucosa in vivo and in vitro. This result is in accordance with results from previous experiments, demonstrating that the level of P120ctn is significantly higher in gastric carcinoma, breast cancer, endometrial cancer, and non-small-cell lung cancer compared with corresponding normal tissues.,,, Based on these results, we postulate that P120ctn is associated with the progression of OSCC.
P120ctn belongs to the catenin family, which also contains α-, β-, and γ-catenin. P120ctn was first reported in 1989 as an Src substrate, and a new member of the catenin family, which contains an armadillo (Arm) repeat domain that directly interacts with cadherins. Unlike the other catenins, P120ctn binds loosely to the JMD of E-cad, forming the cadherin catenin complex (CCC)., Reports have shown the core role of P120ctn is to stabilize cadherin at the cell surface by prohibiting its endocytosis and degradation.,,, Research on P120ctn-deficient SW48 carcinoma cells revealed that the deficiency of P120 causes a reduction in E-cad levels, limiting the adhesiveness and epithelial morphology of these cells. Restoring P120 expression in SW48 cells reactivated E-cad function and rescued an epithelial-like morphology. In our study, there was a significant concordance between the expression of E-cad and P120ctn. Consistent with P120ctn expression, the aberrant expression of E-cad was significantly upregulated in OSCC compared with normal oral mucous mucosa, likely inhibiting migration and differentiation in OSCC.
P120ctn was knocked down and overexpressed in HN4 primary and HN12 metastatic oral cancer cells, respectively. We found the expected correlation between the expression levels of P120ctn and E-cad. The overexpression of P120ctn in HN12 cells increased E-cad expression and decreased the invasion and migration of HN12 cells. In contrast, HN4/shP120ctn cells had increased cell invasion and migration. In addition, our data indicate that P120ctn is critical for EMT in vitro, as shRNA-mediated downregulation of P120ctn expression in HN4 primary cells resulted in an elevation of EMT markers (N-cadherin, vimentin), whereas the overexpression of P120ctn in HN12 metastasis cells induced a profound downregulation of EMT markers. During the development of EMT, a highly co-ordinated and specific series of events defines the transition between epithelial cells and mesenchymal cells. E-cad, which adjusts epithelial cell adhesion, is replaced by N-cadherin, providing more transient adhesive properties which prime the cell for the mesenchymal phenotype. Cytoskeletal elements are reorganized and the peripheral actin cytoskeleton is replaced by stress fibers. In addition, cytokeratin intermediate filaments are replaced by vimentin. Together, these changes shift the cell from a cuboidal to a spindle shape. Finally, the cell acquires the ability to invade and move into the extracellular matrix without any cell–cell contacts. Therefore, P120ctn may inhibit EMT in OSCC by increasing the expression of E-cad. This agrees with results from previous research on breast cancer  and colon carcinoma.
In conclusion, P120ctn may play a key role in the biology of tumor progression. Our data indicate that the overexpression of P120ctn leads to elevated E-cad expression and regulates EMT. Given the effects of P120ctn expression on cell invasion and migration, our data suggest that the proper expression of P120ctn may serve as a therapeutic goal for inhibiting the progression of OSCC.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]
[Table 1], [Table 2], [Table 3]