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Cancer Cell Biology
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Genetic and epigenetic inactivation of T-cadherin in human hepatocellular carcinoma cells
Article first published online: 13 JUN 2008
DOI: 10.1002/ijc.23634
Copyright © 2008 Wiley-Liss, Inc.
Additional Information
How to Cite
Chan, D. W., Lee, J. M.F., Chan, P. C.Y. and Ng, I. O.L. (2008), Genetic and epigenetic inactivation of T-cadherin in human hepatocellular carcinoma cells. Int. J. Cancer, 123: 1043–1052. doi: 10.1002/ijc.23634
Publication History
- Issue published online: 17 JUN 2008
- Article first published online: 13 JUN 2008
- Manuscript Accepted: 14 MAR 2008
- Manuscript Received: 27 NOV 2007
Funded by
- Hong Kong Research Grants Council Central Allocation Grant. Grant Number: HKU 1/06C
- University of Hong Kong (URC)
- Abstract
- Article
- References
- Cited By
Keywords:
- T-cadherin;
- hepatocellular carcinoma cells;
- endothelial cells;
- underexpression;
- LOH;
- hypermethylation;
- c-Jun;
- HCC
Abstract
T-cadherin is an atypical cadherin and growing evidence has indicated that T-cadherin exerts tumor-suppressive effects on cancers of epithelial cell type and also causes positive effects on tumor angiogenesis. Human hepatocellular carcinoma (HCC) is a hypervascular tumor and T-cadherin has been shown to be overexpressed in intratumoral endothelial cells of HCCs. However, the expression status and functions of T-cadherin in hepatocytes or HCC cells remain unclear. Here, we demonstrated that T-cadherin was underexpressed in HCC cells (26.5%, 13/49 cases), but was frequently (77.6%, 38/49) overexpressed in intratumoral endothelial cells immunohistochemically. Semiquantitative RT-PCR analysis also showed that the T-cadherin gene was underexpressed in 7 of 11 HCC cell lines. Loss of heterozygosity analysis revealed that 32–38% of the 42 human HCC samples had allelic losses at this locus. Upon pharmacological treatment with demethylating agent 5-aza-2′-deoxycytidine or histone deacetylase inhibitor trichostatin A, T-cadherin promoter hypermethylation and/or histone deacetylation was frequently observed in HCC samples and cell lines. Functionally, enforced expression of T-cadherin induced G2/M cell cycle arrest, reduced cell proliferation in low serum medium, suppressed anchorage-independent growth in soft agar and increased sensitivity to TNFα-mediated apoptosis in HCC cells. Intriguingly, we found that T-cadherin significantly suppressed the activity of c-Jun, a crucial oncoprotein constitutively activated in HCC cells. To conclude, T-cadherin was differentially expressed in human HCCs. The underexpression of T-cadherin in HCC cells suggests it may be another critical event in addition to T-cadherin-mediated angiogenesis during HCC development. © 2008 Wiley-Liss, Inc.
Hepatocellular carcinoma (HCC) is one of the common malignancies worldwide and is the second leading cause of cancer death in Southeast Asia and Hong Kong.1 Hepatocarcinogenesis is a slow and multistage process involving multiple genetic alterations. These genetic alterations lead to activation of oncogenes and/or inactivation of tumor-suppressor genes.2 The understanding of these molecular mechanisms during the progression of HCC is crucial in developing effective and curative treatments for this cancer.
T-cadherin (CDH13, H-cadherin) is a nonclassical cadherin, which lacks the transmembrane and cytoplasmic domains and is bound to the plasma membrane via a glycosylphosphatidylinositol (GPI) anchor.3 Its protein structure indicates that it may play a role in intracellular signaling and cell–cell adhesion.3, 4 Recent studies have found that T-cadherin is frequently underexpressed in human cancers such as breast, lung, ovary, bladder, colorectal cancers and hematological malignancies and is able to inhibit tumorigenicity.5–10 On the other hand, accumulating evidence has indicated that T-cadherin has positive roles on endothelial and vascular cells during atherosclerosis, neointima formation in experimental restenosis and tumor neovascularization.11–13 These suggest that T-cadherin possesses multiple functions that may be different in different cell types. HCC is typically a hypervascular tumor.14 Recent investigations have found that T-cadherin was overexpressed in intratumoral endothelial cells and played positive roles in angiogenesis of HCC.15, 16 However, the expression status and roles of T-cadherin in HCC cells or hepatocytes remain unclear.
In the present study, we examined the T-cadherin expression in HCC samples immunohistochemically and found that T-cadherin was underexpressed in HCC cells while it was overexpressed in intratumoral endothelial cells. T-cadherin underexpression was further evidenced in HCC cell lines and was associated with frequent allelic losses, promoter hypermethylation and histone deacetylation in both cell lines and HCC samples. We also observed that T-cadherin exerted negative effects on the tumorigenic properties of HCC cells possibly via suppression of c-Jun activity, which is constitutively activated in human cancers and HCC. Altogether, these findings suggest that T-cadherin is differentially expressed in HCC and may exert different functions in hepatocarcinogenesis.
Material and methods
Tumor samples and cell lines
Forty-nine patients (37 male and 12 female; mean age, 49.5 years old; range, 24–74 years old) who had primary HCC resected at Queen Mary Hospital, Hong Kong, between 1992 and 2000, were randomly selected. Thirty-six (73.5%) patients were positive for hepatitis B surface antigen (HBsAg), whereas 7.9% patients were positive for antihepatitis C virus antibody and they were HBsAg negative. All samples were immediately frozen in liquid nitrogen and kept at −80°C until analysis. The nontumorous liver samples were taken at sites away from the tumors. Human hepatoma cell lines, PLC/PRF/5, Hep3B, SMMC7721, BEL7402, SNU368, HepG2, SK-Hep-1, SNU182, SNU449, SNU475 and an immortalized normal liver cell line, LO2, were grown at 37°C in 5% CO2 in MEM or DMEM supplemented with 10% FBS. Transfected cells were selected by G418 at 400 μg/ml for 2 weeks.
Plasmids and cell transfection
The T-cadherin-expressing vector, pcDNA-HA/T-cadherin with haemagglutinin (HA) epitope, was a gift from Dr. Hug C. Lipofectamine™ 2000 (Invitrogen, Tokyo, Japan) was used for cell transfection. The empty pcDNA 3.0 vector was used as mock control.
DNA and RNA extraction
Genomic DNA was extracted by phenol–chloroform after proteinase K treatment. Total RNA was extracted with TRIzol reagent (Invitrogen) and treated with DNaseI (Promega, Madison, WI) according to the manufacturer's instructions. cDNA was synthesized from 2 μg of total RNA using GeneAmp RNA PCR kit components (Applied Biosystems, Foster City, CA).
Reverse transcription-polymerase chain reaction (RT-PCR)
The amount of T-cadherin mRNA was detected by semiquantitative RT-PCR with specific primers [5′-TTCAGCAGAAAGT GTTCCATAT-3′ (forward) and 5′-GTGCATGGACGAACA GAGT-3′ (reverse)] and with 30 cycles of amplification at 94°C for 30 sec, 54°C for 30 sec and 72°C for 30 sec, and was normalized using GAPDH mRNA with the following GAPDH primers [5′-ACGCATTTGGTCGTATTGGG-3′ (forward) and 5′-TGA TTTTGGAGGGATCTCGC-3′ (reverse)] with 25 cycles of amplification at 94°C for 30 sec, 55°C for 30 sec and 72°C for 30 sec.
Treatment with 5-aza-2′ deoxycytidine and trichostatin A
Cells were treated with either of the following drugs: demethylating agent 5-aza-2′-deoxycytidine (5-aza-dC) (Sigma, St Louis, MO) at various concentrations for 4 days, or histone deacetylase inhibitor, trichostatin A (TSA)17 (Sigma), at 500 ng/ml for 1 day. Total RNA was extracted and cDNA synthesized as above.
Sodium bisulfite treatment, bisulfite PCR and sequencing
Sodium bisulfite treatment was carried out using a modified protocol from Clark et al.18 Five micrograms of bisulfite-treated DNA was subjected to PCR using specific primers at T-cadherin 5′-region [5′-GTGATGTTGTTGTTGATTTATTTGG-3′ (forward) (nucleotides −24 to +1), and 5′-AACCCCTCTTCCCTAC CTAAAA-3′ (reverse) (nucleotides +163 to +184)]. PCR reaction was carried out as follows: 94°C for 10 min, 40 cycles of 94°C for 40 sec, 63°C for 30 sec and 72°C for 30 sec, and followed by a final extension step at 72°C for 10 min. Amplified PCR fragments were subcloned into pGEM-T Easy vector (Promega). Bisulfite sequencing was performed on at least 10 individual clones for each cell line.
Analysis of LOH
Because of a lack of choices for microsatellite markers at chromosomal region 16q24.2-3, only 2 microsatellite markers (D16S3091 and D16S402) containing the T-cadherin locus were selected for LOH analysis (Applied Biosystems). Microsatellite markers were amplified from 50–100 ng DNA extracted from HCC and their corresponding nontumorous livers and then analyzed on a model of 377 automatic DNA sequencer (Applied Biosystems), according to the manufacturer's instructions. The results were analyzed with Genotyper software (Applied Biosystems).
Immunohistochemistry and Western blot analysis
Immunohistochemical staining for T-cadherin was performed on formalin-fixed, paraffin-embedded 4-μm-thick sections using the labeled streptavidin-biotin method. The sections were immunostained with rabbit antihuman T-cadherin polyclonal antibody (sc-7940, Santa Cruz Biotechnology, Santa Cruz, CA) in 1:100 dilution. For negative controls, the primary antibody was replaced with Tris-buffered saline. The expression level of T-cadherin analyzed by immunohistochemical staining was scored in the following grades according to the percentage of positive HCC carcinoma cells or hepatocytes: –, <5% positive; +, 5–20% positive; ++, 21–50% positive; +++, >50% positive.
For Western blot analysis, samples containing equal amounts of protein were separated by SDS-PAGE and electroblotted onto Hybond-P membranes (Amersham Pharmacia Biotech, Cleveland, OH). Blots were blotted with 5% skim milk and analyzed by immunoblotting with antibodies specific for HA (Roche Diagnostics, Indianapolis, IN), c-Jun (BD Transduction Laboratories, Franklin Lakes, NJ), phospho-c-Jun (Ser63) II and phospho-JNK (98F2) (Cell Signaling, Beverly, MA). Blots were then incubated with goat antimouse or antirabbit secondary antibodies conjugated to horseradish peroxidase (Amersham) and visualized by enhanced chemiluminescence (ECL) (Amersham).
Luciferase reporter assay
Cells were seeded in 24-well plates and transiently transfected with various amounts of pcDNA-HA/T-cadherin vector with c-Jun PathDetect Trans-Reporting System (pFA-cJun and pFR-luciferase reporter constructs) (Strategene, La Jolla, CA). Luciferase activity was measured using the Dual-luciferase Reporter Assay System (Promega), and transfection efficiency was normalized with Renilla luciferase activity. All experiments were repeated 3 times.
Cell cycle analysis
The cell cycle of cells was determined by fluorescence-activated cell analysis of propidium iodide-stained cells on a FACS Calibur flow cytometer19 (Becton Dickinson, San Jose, CA). Cell cycle analysis was performed using the Multicycle software (Beckman Coulter, Fullerton, CA).
Cell apoptosis analysis
Cells were exposed to the proapoptotic stimulus actinomycin D (ActD, 250 ng/ml) (Sigma) plus TNF-α (Sigma) at various doses (0, 5, 10 and 20 ng/ml) for 24 hr,20 followed by PI immunofluorescence and flow cytometric analysis. The percentage of apoptotic cells was quantified by measurement of the <2 N DNA peak according to Nicoletti et al.21 Terminal deoxynucleotidyltransferase dUTP nick end labeling (TUNEL) analysis for DNA fragmentation22 was carried out using POD cell death detection kit (Boehringer Manheim, Manheim, Germany) according to the manufacturer's instructions. The apoptotic cells were analyzed by TUNEL assay and observed under fluorescence microscopy. Green nuclear staining represents apoptotic nuclei.
Cell proliferation analysis
Cell proliferation rate was measured by Cell Proliferation Kit II (XTT) for 5 days according to the manufacturer's instructions (Roche Diagnostics, Mannheim, Germany). The experiment was performed in triplicate for each time point.
Anchorage-independent growth assay in soft agar
Cells (1 × 104) were trypsinized and suspended in 2 ml of full medium plus 0.3% agar (Sigma). The agar–cell mixture was plated on top of a bottom layer with 1% full medium agar mixture. The experiment was performed in triplicate for each cell line. After 2–3 weeks, viable colonies of 20 cells or more were scored.
Statistical analysis
All data were expressed as mean ± SD for experiments performed at least 3 times. Fisher's exact test was used for the analysis of categorical data. Data were analyzed using the SPSS for Windows 14.0 (SPSS, Chicago, IL). Tests were considered significant when their p values were less than 0.05.
Results
Expression of T-cadherin in human HCC tissues
We first evaluated the levels of T-cadherin transcript in HCC samples with semiquantitative RT-PCR. Of the 49 pairs of human HCC, 28 (57.1%) showed increased T-cadherin expression level by ≥2 folds when compared with the corresponding nontumorous livers (Fig. 1a), and only 5 (10.2%) showed decreased T-cadherin expression level by ≥2 folds. To further investigate the precise expression status of T-cadherin in different cell types, we successfully examined 49 pairs of HCC samples with immunohistochemical staining. Consistent with recent reports,15, 16, 23 Positive T-cadherin immunohistochemical staining was observed in the sinusoidal endothelial cells within the tumors (intratumoral) in 38 (77.6%) of the 49 cases. However, there was no or minimal staining in the sinusoidal endothelial cells in the corresponding nontumorous livers (Fig. 1b) (Table I). The location of the T-cadherin-positive staining in intratumoral endothelial cells was confirmed by immunohistochemical staining using anti-CD34 antibody (data not shown).
Figure 1. Expression analysis of T-cadherin in human HCC. (a) Semiquantitative RT-PCR analysis on T-cadherin expression in tumorous (T) and nontumorous (NT) tissues of representative human HCCs. The graph shows the relative expression of T-cadherin in human HCC samples, with normalization by GAPDH. (b) Immunohistochemical staining showed that T-cadherin was selectively expressed in sinusoidal endothelial cells (arrows) within the HCC tissue when compared with the nontumorous liver tissue. (c) Higher level of T-cadherin expression was detected in the non-neoplastic hepatocytes in the nontumorous liver tissue when compared with HCC cells in the tumorous tissue. (d) T-cadherin expression localized mainly in the cytoplasm and at cell membrane in hepatocytes.
| Case number | T-cadherin expression | LOH result | |||||
|---|---|---|---|---|---|---|---|
| HCC cells | NT-hepatocytes | Expression* | T-endo | NT-endo | D16S3091 | D16S402 | |
| |||||||
| 102 | − | + | Underexpression | + | − | ND | ND |
| 110 | − | + | Underexpression | + | − | LOH | LOH |
| 201 | − | − | = | + | − | NI | R |
| 206 | − | − | = | + | − | R | LOH |
| 208 | + | + | = | + | − | R | R |
| 211 | − | − | = | + | − | R | R |
| 212 | + | − | Overexpression | − | − | R | R |
| 217 | + | + | = | + | − | LOH | LOH |
| 218 | + | − | Overexpression | + | − | NI | R |
| 222 | − | − | = | + | − | LOH | LOH |
| 223 | − | − | = | + | − | ND | ND |
| 228 | + | +++ | Underexpression | − | − | LOH | LOH |
| 229 | − | ++ | Underexpression | − | − | LOH | LOH |
| 230 | − | − | = | − | − | ND | ND |
| 232 | − | − | = | + | − | ND | ND |
| 233 | − | − | = | + | − | NI | LOH |
| 234 | − | + | Underexpression | − | − | R | R |
| 235 | − | − | = | + | − | R | R |
| 240 | − | − | = | + | − | R | R |
| 245 | − | − | = | + | − | ND | ND |
| 247 | + | − | Overexpression | − | − | ND | ND |
| 248 | − | − | = | + | − | LOH | LOH |
| 249 | − | − | = | + | − | R | R |
| 253 | + | + | = | + | − | R | R |
| 254 | − | − | = | + | − | R | R |
| 255 | ++ | ++ | = | − | − | R | R |
| 258 | − | − | = | + | − | R | R |
| 259 | − | − | = | + | − | LOH | LOH |
| 261 | − | + | Underexpression | + | − | ND | ND |
| 264 | − | − | = | − | − | NI | R |
| 266 | − | − | = | + | − | R | R |
| 269 | − | − | = | + | − | R | R |
| 274 | + | − | Overexpression | − | − | LOH | LOH |
| 277 | − | − | = | + | − | R | R |
| 278 | ++ | − | Overexpression | + | − | R | R |
| 280 | − | − | = | − | − | R | NI |
| 284 | − | + | Underexpression | + | − | LOH | LOH |
| 286 | − | − | = | + | − | R | R |
| 291 | − | + | Underexpression | + | − | R | R |
| 292 | − | + | Underexpression | + | − | R | R |
| 294 | − | − | = | + | − | R | LOH |
| 296 | − | + | Underexpression | − | − | R | R |
| 304 | − | ++ | Underexpression | + | − | R | R |
| 309 | − | + | Underexpression | + | − | LOH | LOH |
| 310 | − | − | = | + | − | R | R |
| 312 | − | − | = | + | − | NI | R |
| 315 | − | − | = | + | − | R | LOH |
| 316 | − | − | = | + | − | LOH | LOH |
| 320 | − | ++ | Underexpression | + | − | LOH | LOH |
Intriguingly, we observed that 13 of 49 cases (26.5%) showed either loss or underexpression of T-cadherin in the HCC cells immunohistochemically, when compared with the non-neoplastic hepatocytes in the corresponding nontumorous livers (Table I). It is to note that all these 13 cases showed T-cadherin overexpression with semiquantitative RT-PCR analysis, likely as a result of overexpression of T-cadherin in the intratumoral endothelial cells. For the remaining cases, only 5 (10.2%) had higher T-cadherin expression levels in the HCC cells and the other 31 (63.3%) had no significant difference in the expression levels between HCC cells and their corresponding non-neoplastic hepatocytes (Fig. 1c) (Table I). The staining pattern for T-cadherin was patchy in both tumorous and nontumorous tissues, and T-cadherin was localized in the cytoplasm and at the cell membranes of HCC cells and non-neoplastic hepatocytes (Figs. 1c and 1d). Taking together, these data indicate that T-cadherin is differentially expressed in the 2 cell types within HCC samples. The underexpression of T-cadherin in HCC cells and overexpression of T-cadherin in intratumoral endothelial cells suggest that T-cadherin may have different functions in these 2 cell types in hepatocarcinogenesis.
Upon clinicopathological correlation, T-cadherin underexpression in HCCs was significantly associated with positive hepatitis B surface antigen (HBsAg) status (p = 0.046) (Table II). However, there was no association between underexpression of T-cadherin and tumor size, encapsulation, cellular differentiation, number of tumor nodules, presence of venous invasion, tumor microsatellite formation, cirrhosis and tumor stage. Overexpression of T-cadherin in intratumoral endothelial cells immunohistochemically, however, had no significant association with any of clinicopathological parameters analyzed.
| Parameters | Number of cases (%) | P value | |
|---|---|---|---|
| Underexpression of T-cadherin | Normal/overexpression of T-cadherin | ||
| |||
| Tumor size | |||
| <5 cm | 4 (30.8) | 15 (41.7) | 0.741 |
| >5 cm | 9 (69.2) | 21 (58.3) | |
| Cellular differentiation (Edmondson's grading) | |||
| Grade I–II | 4 (30.8) | 18 (50.0) | 0.333 |
| Grade III–IV | 9 (69.2) | 18 (50.0) | |
| Tumor encapsulation | |||
| Absent | 8 (61.5) | 26 (74.3) | 0.480 |
| Present | 5 (38.5) | 9 (25.7) | |
| Tumor microsatellite formation | |||
| Absent | 6 (46.2) | 17 (47.2) | 1.000 |
| Present | 7 (53.8) | 19 (52.8) | |
| Venous invasion | |||
| Absent | 6 (46.2) | 16 (44.4) | 1.000 |
| Present | 7 (53.8) | 20 (55.6) | |
| Number of tumor nodules | |||
| 1 | 12 (92.3) | 29 (80.6) | 0.663 |
| ≥2 | 1 (7.7) | 7 (19.4) | |
| Cirrhosis | |||
| Absent | 8 (61.5) | 21 (58.3) | 1.000 |
| Present | 5 (38.5) | 15 (41.7) | |
| Tumor stage (pTNM) | |||
| Stage I–II | 4 (33.3) | 12 (35.3) | 1.000 |
| Stage III–IV | 8 (66.7) | 22 (64.7) | |
| Serum hepatitis B surface antigen | |||
| Positive | 12 (100) | 25 (71.4) | 0.046* |
| Negative | 0 (0) | 10 (28.6) | |
Allelic loss of T-cadherin in human HCC
The 16q24 chromosomal region is a frequent deletion site in human cancers, including HCC.24 Therefore, we analyzed the allelic losses with LOH assay using 2 microsatellite markers (D16S3091 and D16S402) flanking the T-cadherin locus. Of the 49 HCC cases, 42 were successfully examined for LOH at the D16S3091 and D16S402 loci, and LOH was found in 32.4% (12 of 37 informative cases) and 37.5% (15 of 40 informative cases) (Fig. 2) (Table I). Among these 42 HCC cases, 6 (14.3%) cases had both LOH and underexpression of T-cadherin in HCC cells and 19 (45.2%) cases had neither allelic losses nor difference in T-cadherin expression levels between tumorous and nontumorous tissues. Seven cases (16.7%) showed no allelic losses but underexpression of T-cadherin, and 10 (23.8%) cases showed allelic losses but had either no difference in T-cadherin expression (9 cases) or overexpression (1 case).
Figure 2. Allelic losses of T-cadherin locus in human HCC. A schematic map showing the T-cadherin locus and the positions of the 2 microsatellite markers used for LOH analysis. A representative case of human HCC showing allelic losses at the 2 microsatellite markers. Arrows point to the deleted alleles in tumor DNA.
Silencing of T-cadherin by hypermethylation and histone deacetylation in HCC
With semi-quantitative RT-PCR analysis, 7 of 11 hepatoma cell lines (HepG2, Hep3B, SNU182, HLE, HUH7, PLC/PRF/5 and SNU449) showed either loss or underexpression of T-cadherin when compared with an immortalized normal liver cell line, LO2 (Fig. 3a).
Figure 3. T-cadherin silencing by hypermethylation and histone deacetylation (a) T-cadherin mRNA expression in human hepatoma cell lines. (b) Restoration of the expression of T-cadherin after treatment with 5-aza-dC. (c) A schematic diagram showing the 5′ region of the T-cadherin gene and location and methylation status of CpG dinucleotides in bisulfite sequencing region. The positions of the CpG dinucleotides in the genomic sequence (GenBank accession number AB001090) are indicated by thin vertical lines. The bent arrow indicates the transcription start site (+1), and the arrowhead the translation start site (TSS). The horizontal open bar indicates the region of the bisulfite amplicon. Filled circles represent methylated dinucleotides and open circles represent nonmethylated dinucleotides. Ten clones were subjected to sequencing and the clonal numbers are indicated by the prefix C on the left. The percentage on the right indicates the extent of methylated CpG dinucleotides in each cell line. (d) The expression of T-cadherin was restored after treatment with TSA and detected by semiquantitative RT-PCR.
To evaluate the relationship between the methylation status in T-cadherin promoter region and T-cadherin expression levels, we treated 7 HCC cell lines with the demethylating agent 5-aza-dC at various doses for 4 days. With semiquantitative RT-PCR analysis, the expression of T-cadherin was restored in SNU449 at 0.5 μM, in SNU182 at 0.1 μM and in HLE at 10 μM of 5-aza-dC (Fig. 3b) (Table III).
| HCC cell lines with low or no T-cadherin expression | HBV/HCV | Epigenetic mechanisms | |
|---|---|---|---|
| Hypermethylation | Histone deacetylation | ||
| |||
| HepG2 | − | − | + |
| Hep3B | HBV-positive | − | − |
| HLE | − | + | + |
| HUH7 | − | − | + |
| PLC/PRF/5 | HBV-positive | − | − |
| SNU182 | − | + | − |
| SNU449 | − | + | + |
We further evaluated the methylation status of CpG islands in T-cadherin promoter region in these HCC cell lines by bisulfite sequencing analysis. Of the 7 HCC cell lines with low or no T-cadherin expression, SNU182 and SNU449 showed significantly higher level of methylation of CpG dinucleotides when compared with SMMC7721, which expressed relatively higher level of T-cadherin (Fig. 3c). HLE had mild level of methylation of the CpG islands in the T-cadherin promoter region (Fig. 3b). The remaining 4 HCC cell lines (HepG2, Hep3B, HUH7 and PLC/PRF/5) had similar methylation status as SMMC7721 (data not shown). We also successfully performed bisulfite sequencing analysis on 2 representative HCC samples with either underexpression (Cases 320 and 291) or no difference in T-cadherin expression between tumorous and nontumorous tissues (Case 310) (Table I). Consistent with the immunohistochemical results, Case 320 showed significantly higher level of methylation of CpG dinucleotides in the T-cadherin promoter region of tumorous tissue (62%) when compared with the corresponding nontumorous tissue (20%), whilst Case 310 showed no difference in methylation status between the tumorous and nontumorous tissues (Fig. 3b). It is of note that Case 320 had LOH at both D16S3091 and D16S402 loci, whereas Case 310 had retention at both loci. However, Case 291, which had lower expression level of T-cadherin in the tumorous tissues as demonstrated with IHC analysis, showed relatively low level of methylation status of the CpG dinucleotides in both tumors and nontumorous tissues (Fig. 3b). There was, however, no LOH at both loci. This indicates that there may be mechanisms other than DNA methylation accounting for T-cadherin gene silencing.
To evaluate if histone deacetylation also played a role in T-cadherin gene silencing, we treated these 7 HCC cell lines with TSA. Semiquantitative RT-CR analysis revealed that 4 HCC cell lines (HepG2, HLE, HUH7 and SNU449) had re-expression of T-cadherin after treatment with TSA (Fig. 3d) (Table III).
Collectively, these data suggest that in addition to allelic loss at the T-cadherin locus, hypermethylation and histone deacetylation also contribute to T-cadherin silencing in HCC.
T-cadherin induced G2/M cell cycle arrest
To investigate the functions of T-cadherin in HCC cells, T-cadherin stably expressing clones were established in PLC/PRF/5 cells, in which the expression of T-cadherin was relatively low (Fig. 4a). Two stable clones (C5 and C8) of PLC/PRF/5 with different expression levels of T-cadherin were established, and the pcDNA empty vector transfectant was used as the mock control (Fig. 4a). Since previous studies have shown that T-cadherin induced G2/M arrest in gliomas and cutaneous squamous carcinoma,26, 27 we examined the cell cycle changes of T-cadherin-stable transfectants with flow cytometric analysis. After cell synchronization with serum-free treatment, we found that all T-cadherin transfectants of PLC/PRF/5 (C5 and C8) had higher proportions of cells at the G2/M phase than their vector controls (Fig. 4b), and the increase was consistent with the expression levels of T-cadherin. These findings indicate that T-cadherin induced G2/M arrest in these cells in a dose-dependent manner.
Figure 4. T-cadherin induced G2/M arrest in HCC cells. (a) Western blot analysis showing the expression of T-cadherin in PLC/PRF/5 and stable transfectants. (b) A representative flow cytometric profile and a bar graph showing the corresponding cell cycle distribution in percentage of the T-cadherin-stable transfectants and vector control (M).
T-cadherin suppressed HCC cell proliferation and anchorage-independent growth
With XTT cell proliferation assay, no obvious difference was observed in the cell proliferation rates between the T-cadherin-stable clones and the mock controls when grown in normal culture medium (10% FCS) (data not shown). In contrast, when these clones were cultured in low serum medium (0.1% FCS), there was a remarkable reduction in the cell proliferation rates of the T-cadherin transfectants (C5 and C8 of PLC/PRF/5 cells) when compared with their mock controls (Fig. 5a). This suggests that T-cadherin inhibits cell growth of HCC cells when they are subjected to extracellular stress condition.
Figure 5. T-cadherin inhibited cell proliferation and anchorage-independent growth of HCC cells. (a) XTT assay demonstrated that T-cadherin-stable transfectants had slower proliferation rates when compared with vector controls in PLC/PRF/5 cells when cultured in low serum medium, *p = 0.004 for PLC/PRF/5 C5 and C8 when compared with the mock control. (b) The soft agar assay showed a significant reduction in the anchorage-independent growth ability of T-cadherin-stable transfectants (C5 and C8 of PLC/PRF/5), when compared with their mock controls. *p < 0.001.
To further investigate the effects of T-cadherin on tumorigenicity of HCC cells, we assessed the anchorage-independent growth ability in soft agar of the T-cadherin-stable clones and mock control. At 2 weeks, the vector control of PLC/PRF/5 cells had approximately a mean of 1,259 colonies (Fig. 5b). In contrast, their T-cadherin-stable transfectants (C5 and C8) showed a dramatic reduction in the number of the colonies in soft agar (mean of 331 and 780 colonies) (Fig. 5b). These data indicate that enforced expression of T-cadherin impairs anchorage-independent growth in HCC cells.
T-cadherin sensitized HCC cells to TNFα-induced apoptosis
Since loss of susceptibility to apoptosis is a crucial factor in carcinogenesis, we sought to test whether T-cadherin could also sensitize HCC cells to TNFα-induced apoptosis. With flow cytometric analysis, we found that the T-cadherin-stable transfectants showed significantly higher apoptotic rates (C5 and C8 of PLC/PRF/5 cells) when compared with their mock controls, in a dose-dependent manner and consistent with the expression levels of T-cadherin in these stable clones (Fig. 6a).
Figure 6. T-cadherin sensitized TNFα-induced apoptosis in HCC cells. (a) PLC/PRF/5 T-cadherin-stable transfectants and vector control (M) clones were treated with TNFα at increasing doses. The percentage of sub-G1 events (apoptotic cells) of each clone was measured by flow cytometric analysis. *p < 0.001 when compared with their mock controls. (b) PLC/PRF/5 T-cadherin-stable transfectants, and vector control clones were treated with TNFα at 20 ng/ml and examined by TUNEL assay.
The TUNEL assay further confirmed that there were more apoptotic cells in the T-cadherin clones (C5 and C8 of PLC/PRF/5 cells) when compared with the vector controls (Fig. 6b). These results suggest that T-cadherin may sensitize HCC cells to apoptosis.
T-cadherin suppressed c-Jun activity
To investigate the molecular mechanisms of T-cadherin in relation to the phenotypic effects of HCC, we tested a few common signaling pathways such as c-Jun by PathDetect Trans-Reporting Systems (Strategene), NF-κB by PathDetect Cis-Reporting system (Strategene), Wnt/β-catenin pathway by TOP/FOP system and Stat3 by Western blot analysis in HCC cells (data not shown). Intriguingly, in HCC cells, T-cadherin had inhibitory effect solely on c-Jun activity. Upon transfection of T-cadherin at increasing amounts (0, 250 and 500 ng), the relative luciferase activity of c-Jun was reduced from 100% to 80 and 57%, respectively, in HepG2 cells (P <0.001), from 100% to 50 and 28%, respectively, in PLC/PRF/5 cells (P <0.001), and from 100% to 87 and 70%, respectively, in BEL7402 cells (p < 0.001) (Fig. 7a). Despite such a difference in the degree of inhibitory effect on c-Jun by T-cadherin among the HCC cell lines, the data suggest that T-cadherin is capable of suppressing c-Jun activity in human HCC cells.
Figure 7. T-cadherin suppressed c-Jun activity via inhibition of c-Jun phosphorylation in HCC cells. (a) The c-Jun Trans-Reporting System demonstrated that the c-Jun-dependent luciferase activity was inhibited by T-cadherin in HepG2, PLC/PRF/5 and BEL7402 cells. *p < 0.001. (b) Western blot analysis of phospho-c-Jun and c-Jun expression levels in T-cadherin-stable transfectants of PLC/PRF/5 cells. (c) Western blot analysis showing the change in phospho-JNK levels in PLC/PRF/5 when stably transfected with HA/T-cadherin. β-Actin was used as loading control. M represents vector control.
We next evaluated the expression levels of phospho-c-Jun in T-cadherin-stable transfectants. Because of the low baseline phosphorylation level of c-Jun (data not shown), HCC cells were stimulated by the alkylating agent methyl methane sulfonate (MMS), which is a potent inducer of the JNK pathway.28 With Western blot analysis, all T-cadherin clones (C5 and C8 of PLC/PRF/5) had reduced phospho-c-Jun and total c-Jun levels compared with their mock controls (Fig. 7b). As the phosphorylation of c-Jun is induced by activated JNK, we evaluated the active form of JNK in HCC cells transfected with T-cadherin. As expected, the levels of phospho-JNK stimulated by MMS were remarkably suppressed in PLC/PRF/5 cells transiently transfected with T-cadherin-expressing vector (Fig. 7c), suggesting that T-cadherin suppresses c-Jun activity via inhibition JNK signalling pathway in HCC cells.
Discussion
Recent studies have shown that T-cadherin was overexpressed in HCC when compared with normal livers or nontumorous liver tissues, and its expression was restricted to intratumoral endothelial cells.15, 16, 29 Also, accumulating evidence has shown that T-cadherin may act as a positive regulator in endothelial cells and be involved in vascularization by facilitating cell cycle progression and promoting cell proliferation of intratumoral vascular cells.13, 15 However, the expression status and functions of T-cadherin in HCC cells or hepatocytes are unclear. In the present study, we employed immunohistochemical analysis to precisely delineate the distribution and expression pattern of T-cadherin in HCC. Similar to the recent reports,15, 16 T-cadherin was highly expressed in the sinusoidal endothelial cells within the tumors (intratumoral) but not in the sinusoidal endothelial cells in the corresponding nontumorous livers. This finding is in accordance with those of the previous reports that T-cadherin is a marker of tumor angiogenesis and plays crucial role during HCC progression.15, 30 On the other hand, we also found that 26.5% of our HCC cases showed either loss or underexpression of T-cadherin in the HCC cells in the tumorous tissues when compared with the non-neoplastic hepatocytes in the corresponding nontumorous livers. This observation contrasts with the results of Adachi et al.15 and Riou et al.,23 who showed either undetectable or overexpression of T-cadherin in HCC, respectively. The discrepancy may be due to the sensitivity of IHC examination. Besides, the difference in the numbers of cases and etiologies of HCC might contribute to this discrepancy. We used more HCC samples for IHC analysis and most of them were HBV positive, while the HCC specimens examined by Riou et al.23 were associated with alcohol consumption. This was evidenced by our clinicopathological data that there was a significant correlation between underexpression of T-cadherin and the presence of HBV infection.
T-cadherin underexpression in HCC cells was further supported by the findings of loss or underexpression of T-cadherin mRNA levels in 7 of 11 HCC cell lines. Moreover, the underexpression of T-cadherin in HCC cell lines and human HCC samples was associated with hypermethylation of T-cadherin promoter. This is in accordance with the findings of hypermethylation of T-cadherin promoter in other human cancers.31–35 More importantly, histone deacetylation, which has not been reported before, was also frequently observed in the hepatoma cell lines with relatively low T-cadherin expression levels. In addition, chromosome 16q24.2–24.3 is one of the frequent regions of allelic losses in human HCC.24, 36T-cadherin is located in this region and has been documented to be underexpressed in a number of human cancers.6–10 The high correlation between LOH results and T-cadherin expression status (59.5%) suggests that allelic loss of T-cadherin may also be a crucial mechanism in the silencing of T-cadherin expression in HCC.
In this and other previous reports, it was found that the T-cadherin mRNA level was upregulated in HCCs using either semiquantitative or real-time quantitative RT-PCR.16, 29 However, the present findings demonstrate that T-cadherin had differential expression in different cell types in HCC. The silenced expression of T-cadherin in HCC cells could have been easily masked by the frequently high expression of T-cadherin in the intratumoral endothelial cells. Thus, RT-PCR without microdissection of the cancer cells may not be able to evaluate the true expression of T-cadherin in HCC cells in human samples.
Functionally, the loss of T-cadherin expression in HCC cells suggests that it may play a suppressive role in tumorigenesis of HCC cells. In this study, we demonstrated that T-cadherin had antiproliferative effects and antitumorigenic roles in HCC cells. These findings were consistent with those of previous studies on other cancer cell types5, 26, 27 and suggest that T-cadherin may have different and perhaps opposite roles in epithelial and endothelial cells. Although the antitumorigenic roles of T-cadherin have been documented in a number of human cancer cell types,5, 26, 27 the molecular mechanisms are unclear. In this study, we have found that T-cadherin had significant inhibition on c-Jun activity in HCC cells. Besides, T-cadherin not only could inhibit JNK and c-Jun activities, it could also induce G2/M cell cycle arrest, inhibit cell proliferation and sensitize HCC cells to TNFα-induced apoptosis. These effects were similar to those of the JNK inhibitor, SP600125,37, 38 suggesting that T-cadherin was a negative regulator to JNK signalling pathway by suppressing JNK phosphorylation. To the best of our knowledge, this is the first report suggesting this function of T-cadherin, and further studies to investigate the roles of T-cadherin in JNK signalling pathway are warranted.
In conclusion, we have shown that T-cadherin was differentially expressed in HCC cells and intratumoral endothelial cells in human HCC samples. The underexpression of T-cadherin in HCC cells might be attributed to frequent allelic losses, promoter hypermethylation and histone deacetylation. Similar to the antitumorigenic effects of T-cadherin in other human cancers,5, 6, 27 T-cadherin was able to inhibit cell growth, enhance apoptosis and suppress tumorigenicity in HCC cells. This antitumorigenic effect may be due to the inhibitory ability of T-cadherin on JNK and c-Jun activities in HCC cells. Overall, these findings suggest that the loss of T-cadherin in HCC cells may be another critical event in addition to T-cadherin-mediated angiogenesis during HCC progression.
Acknowledgements
We wish to thank Dr. C. Hug (Whitehead Institute for Biomedical Research, Massachusetts Institute of Technology, USA) for the pcDNA-HA/T-cadherin-expressing construct and comments.
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