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Molecular Oncology, Markers, Clinical Correlates |
1 Department of Oral Maxillofacial Pathobiology, Division of Frontier Medical Science, Graduate School of Biomedical Sciences, Hiroshima University, Hiroshima, and 2 Center of Oral Clinical Examination, Hiroshima University Hospital, Hiroshima, Japan
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ABSTRACT |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The cell adhesion molecule E-cadherin is a transmembrane glycoprotein responsible for homotypic binding and morphogenesis of epithelial tissues (4 , 5) . E-cadherin is arguably the prototypic member of the classical cadherin family and plays a critical role in cell-to-cell adhesion. The cytoplasmic domain of E-cadherin is linked to the actin cytoskeleton through critical interactions with catenins (6) . Disruption of the cadherin-catenin complex has been demonstrated in various cancers and has been correlated with tumor differentiation, invasion, metastasis, and prognosis. Immunohistochemical studies have demonstrated that loss of E-cadherin expression is a frequent event in various types of cancers (6) . In OSCC, E-cadherin also correlates well with invasion and metastasis (7, 8, 9, 10) . However, mutations of the CDH1 gene encoding E-cadherin are rare or absent, and CDH1 gene mutations that compromise the nonadhesive function of E-cadherin have been observed only in human gastric carcinoma cell lines, lobular breast cancer, and familial gastric cancer (11) . In addition, loss of E-cadherin expression is heterogeneous in cancers and may be modulated by the tumor microenvironment (11) . These results suggest that the mechanism of E-cadherin loss in these tumors does not involve irreversible genetic alterations. Methylation associated with E-cadherin loss in breast cancers is heterogeneous and unstable (12) . Proposed epigenetic mechanisms for E- cadherin loss include alterations in the expression and/or function of trans-acting factors that regulate CDH1 gene transcription, hypermethylation of the CDH1 promoter, and chromatin-mediated effects. Such epigenetic plasticity may contribute to the dynamic, phenotypic heterogeneity that drives metastatic progression.
In adherens junctions, ß-catenin links E-cadherin and the actin cytoskeleton through interaction with 
-catenin (6)
. A human signet ring gastric cancer cell line has a homozygous deletion in the ß-catenin gene CTNNB1, which results in impaired cell-to-cell adhesion (13)
. In addition to its critical role in cellular adhesion, ß-catenin functions in the Wnt signaling pathway (14)
. Consistent with its ostensibly independent functions in cell adhesion and signal transduction, at least two distinct pools of ß-catenin are supposed to exist in cells: a cell membrane-associated pool, and a pool involved in Wnt signaling and gene transcription. In Wnt signaling, ß-catenin is regulated in part by the adenomatous polyposis coli (APC) protein AXIN and glycogen synthase kinase (GSK) 3ß (14)
. A pool of ß-catenin involved in Wnt signaling and gene transcription is degraded by the ubiquitin-proteasome pathway. In the absence of a Wnt signal, ß-catenin is phosphorylated by a multimolecule complex consisting of GSK3, APC, and AXIN, and then the phosphorylated ß-catenin is recognized and ubiquitinated by the E3 ubiquitin ligase complex SCFß-Trcp (15)
. Activation of the Wnt signaling pathway leads to inhibition of ß-catenin degradation by decreasing the ability of GSK3ß to phosphorylate ß-catenin, and stabilization of ß-catenin enhances the interaction with members of the T-cell factor/lymphoid enhancer factor family of transcription factors (16)
. Numerous studies have suggested that ß-catenin is a potent oncogene product, and its accumulation has been implicated in tumorigenesis in various cancers (14)
. In contrast, decreased expression of ß-catenin is found in esophageal, colon, gastric, and oral cancers (17, 18, 19)
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In the present study, we isolated highly invasive clones (MSCC-Inv1 and MSCC-Inv2) from MSCC-1 cells using an in vitro invasion assay and found E-cadherin methylation and ß-catenin degradation in these highly invasive clones. Moreover, we also found frequent E-cadherin methylation and reduced expression of membranous ß-catenin in the invasive and metastatic areas of OSCCs.
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MATERIALS AND METHODS |
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Antibodies and Reagents.
Sources of antibodies and reagents are as follows: Z-Leu-Leu-Leu-CHO (ZLLL), Peptide Institute (Osaka, Japan); cycloheximide, Sigma; mouse anti-E- cadherin antibody (C20820), mouse anti-ß-catenin antibody (C19220), mouse anti-p27 antibody (K25020), and mouse anti-Skp1 antibody (P46020), Transduction Laboratories; rabbit anti-I
B antibody (C-21), Santa Cruz Biotechnology; mouse anti-ß-actin antibody, Sigma; and mouse anti-E-cadherin antibody (clone HECD-1), Takara Bio Inc. (Otsu, Japan).
Tissue Samples.
Tissue samples of OSCCs were retrieved from the Surgical Pathology Registry of Hiroshima University Dental Hospital from 1998 to 2003. Tissues were fixed in 10% buffered-formalin and embedded in paraffin.
In vitro Invasion Assay.
In vitro invasion assay was performed as described previously (20)
. Cells were placed in the upper compartment of the cell culture insert for 6 h. After incubation, we collected cells that had migrated to the lower side of the filter to isolate highly invasive clones by the method of Kalebic et al. (21)
, with minor modification. To examine invasiveness, cells were fixed with methyl alcohol and stained with H&E at the end of the incubation. The invasiveness of the cells was determined by counting the number of cells that migrated through the pores to the lower side of the filter under a microscope at x100 magnification. We performed the assay three times, and three randomly selected fields were counted for each assay. We used Student’s t test to determine the significance of invasive cells by in vitro invasion assay. P <0.05 was required for significance.
Three-Dimensional Culture.
Human dermal fibroblasts were mixed into neutralized type I collagen gel (Cellmatrix type I-A; Nitta Gelatin Inc., Osaka, Japan), and the mixed solution (3 ml/well in 6-well plates) was hardened for 30 min. Then, 2 x 106 cells in 2 ml of three-dimensional culture medium [1:1 mixture of Keratinocyte-SFM (Invitrogen) and Dulbecco’s modified Eagle’s medium supplemented with 10 µg/ml gentamicin and 10% fetal bovine serum] were placed on each gel and incubated overnight. The following day, we detached the hardened gels from the plate. The gels were incubated for 1 week until the size of the contracted gel had plateaued. Cell strainers (Becton Dickinson) were placed upside-down in new 6-well plates after cutting the handle, and three-dimensional culture medium was poured until the nylon mesh of cell strainers was beneath the fluid surface. We then placed the contracted-gel discs on the nylon mesh and added three-dimensional culture medium until the fluid level reached the upper edge of the gel. It is important that the gel is steeped sufficiently in the fluid but that the surface of the gel is dry. The gels were incubated under air-liquid interface culture for 1 more week. During this time, the three-dimensional medium was changed every other day. After this incubation, the gel discs were fixed in phosphate-buffered formalin and embedded in paraffin, and sagittal sections were stained with H&E.
Preparation of Membrane Fraction.
To obtain the membrane fraction, we used the Mem-Per Eukaryotic Membrane Protein Extraction Kit (Pierce). Cells were lysed according to the manufacturer’s instructions.
Western Blot Analysis.
Cells were lysed in cold lysis buffer containing 50 mM Tris-HCl (pH 7.5), 250 mM NaCl, 0.1% Triton X-100 (Roche), 1 mM EDTA, 50 mM NaF, 0.1 mM Na3VO4, 1 mM dithiothreitol, 0.1 mM leupeptin, 0.1 µg/ml soybean trypsin inhibitor, 10 µg/ml L-1 chlor-3-(4-tosylamido)-4-phenyl-2-butanon, 10 µg/ml L-1 chlor-3-(4-tosylamido)-7-amino-2-heptanon-hydrochloride, 10 µg/ml aprotinin, and 50 µg/ml phenylmethylsulfonyl fluoride. Lysates were incubated on ice for 30 min and spun down at 15,000 rpm for 20 min at 4°C. The protein concentration was determined by Bradford protein assay (Bio-Rad) using bovine serum albumin (Sigma) as a standard. Western blot analysis was performed as described previously (22)
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Immunohistochemistry.
Immunohistochemical detection of E-cadherin and ß-catenin in OSCCs was performed on 4.5-µm sections mounted on silicon-coated glass slides, using a streptavidin-biotin peroxidase technique as described previously (22)
. For immunohistochemical detection of ß-catenin in cells, cells were plated on coverslips and fixed with formalin for 30 min after treatment, and then immunohistochemistry was performed as described above.
Laser Capture Microdissection and DNA Extraction.
Ten-micrometer sections mounted on glass slides covered with PEN foil (Leica Microsystems) were used for DNA extraction. The sections were stained slightly with H&E and air-dried. The areas showing no expression or reduced expression of E-cadherin were selected by comparison with the immunostained sections. The selected areas were dissected from the sections with Laser Microdissection systems (Leica Microsystems) and dropped immediately into a microcentrifuge tube cap filled with 30 µl of ATL buffer (DNeasy Tissue Kit; Qiagen), and then DNA was extracted by overnight incubation with proteinase K at 55°C according to the manufacturer’s instructions.
Methylation-Specific PCR for E-cadherin.
Methylation-specific PCR (MSP) primers spanning the transcription start site of E-cadherin were reported previously as island 3 by Graff et al. (12)
. The sets of primers were E-cadherin M-sense (5'-TTAGGTTAGAGGGTTATCGCGT-3') and E-cadherin M-antisense (5'-TAACTAAAAATTCACCTACCGAC-3') for the methylated sequence, and E-cadherin U-sense (5'-TAATTTTAGGTTAGAGGGTTATTGT-3') and E-cadherin U-antisense (5'-CACAACCAATCAACAACACA-3') for the unmethylated sequence. DNA was isolated from cells by using the DNeasy kit (Qiagen). DNA was isolated from paraffin block sections of OSCCs as described above. DNA was denatured by NaOH (final concentration, 0.2 M) for 10 min at 37°C. Thirty microliters of 10 mM hydroquinone (Sigma) and 520 µl of 3 M sodium bisulfite (Sigma) at pH 5.0, both freshly prepared, were added and mixed, and samples were incubated at 50°C for 16 h. Modified DNA was purified using Wizard DNA purification resin (Promega) and eluted into 50 µl of water. Modification was completed by NaOH (final concentration, 0.3 M) treatment for 5 min at room temperature, followed by ethanol precipitation. Modified DNAs were amplified in a total volume of 20 µl of 1x GeneAmp PCR Gold Buffer (PE Applied Biosystems) containing 1.0 mM MgCl2, 1 mM each primer, 0.2 mM deoxynucleoside triphosphates, and 1 unit of Taq polymerase (AmpliTaq Gold DNA polymerase; PE Applied Biosystems). After activation of the Taq polymerase at 95°C for 10 min, PCR was performed in a thermal cycler for 40 cycles consisting of denaturation at 95°C for 30 s, annealing at 57°C (E-cadherin M-primer sets) or 53°C (E-cadherin U-primer sets) for 30 s, and extension at 72°C for 30 s, followed by a final 10-min extension at 72°C. PCR products were then loaded onto nondenaturing 12% polyacrylamide gels, stained with ethidium bromide, and visualized under UV illumination.
Silencing by Small Interfering RNA.
Logarithmically growing HeLa cells were seeded at a density of 105 cells/6-cm dish and transfected twice with oligonucleotides (at 24 and 48 h after replating) using Oligofectamine (Invitrogen) as described previously (23)
. Forty-eight hours after the last transfection, lysates were prepared and analyzed by SDS-PAGE and immunoblotting. The small interfering RNA (siRNA) oligonucleotides used for both ß-Trcp1 and ß-Trcp2 silencing were 21-bp synthetic molecules (Dharmacon Research) corresponding to nucleotides 407–427 of human ß-Trcp1 and nucleotides 161–181 of human ß-Trcp2 (AB033279; Ref. 24
). A 21-nucleotide siRNA duplex corresponding to a nonrelevant F-box protein (Fbp) gene was used as control.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Cell growth of these clones was the same as that of the parent cells (data not shown), but the number of MSCC-Inv1 and MSCC-Inv2 cells that migrated through the filter at 6 h was significantly higher than that of parent cells (P < 0.01; Fig. 1B
). Moreover, we demonstrated that MSCC-Inv1 and MSCC-Inv2 cells had higher invasiveness than parent cells by using three-dimensional culture analysis (Fig. 1C)
. To determine the mechanism responsible for the higher invasiveness in MSCC-Inv1 and MSCC-Inv2 cells, we examined the expression of cell adhesion molecule E-cadherin and ß-catenin in these cells. MSCC-Inv1 and MSCC-Inv2 cells showed reduced expression of E-cadherin and ß-catenin in comparison with parent cells (Fig. 1D)
. A greater reduction of E-cadherin and ß-catenin protein was seen in MSCC-Inv1 cells than in MSCC-Inv2 cells.
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. Control antibody did not enhance the invasiveness of any of the cells. Next, we transfected the E-cadherin gene into parent MSCC-1 cells and MSCC-Inv1 cells and examined the effects on invasion. E-cadherin transfection suppressed the invasiveness of these cells (Fig. 2B)
. The effect on suppression of invasion was greater in MSCC-Inv1 cells than in parent cells. Original MSCC-Inv1 cells did not attach to each other, but E-cadherin-transfected cells showed cell-to-cell adhesion (Fig. 2C)
. These data indicate that loss of E-cadherin plays an important role in invasion through loss of cell-to-cell adhesion in OSCC cells.
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. In MSCC-Inv1 cells, an obvious methylated band was detected in comparison with MSCC-Inv2 cells. Then, we examined E-cadherin promoter methylation in OSCCs by MSP. Two highly invasive OSCCs and 18 lymph node metastases of OSCC were examined. Highly invasive cases (tumors were surgically resected from tongue) showed that cancer cells invaded the underlying muscle. We defined a mucosal or submucosal area without invasion of the underlying muscle as a "noninvasive area." In contrast, "invasive area" was defined as a submucosal area with invasion of the underlying muscle. All metastatic OSCCs were surgically resected from the cervical or submandibular lymph node. At first, we examined the immunoexpression of E-cadherin in paraffin-embedded tissues from these cases. In the two highly invasive OSCCs, OSCC cells in invasive areas showed reduced expression of E-cadherin, whereas OSCC cells in noninvasive areas showed high expression of E-cadherin in their cell membrane (Fig. 3B)
. All 18 metastatic OSCCs showed reduced expression of E-cadherin in nodal lesions, but expression of E-cadherin was heterogeneous (Fig. 3C)
. In these tumors, we asked whether the reduced expression of E-cadherin was caused by methylation or not. Because the pattern of E-cadherin expression in OSCC cases exhibited striking heterogeneity, we examined the methylation of E-cadherin only in microdissected OSCC cells that showed reduced expression of E-cadherin by using the laser capture microdissection technique. In the highly invasive OSCC cases, methylation of OSCC cells was detected in the invasive areas, but not in the noninvasive areas (Fig. 3B
; Table 1
). Interestingly, methylation was seen in 13 of 18 metastatic OSCCs that showed reduced E-cadherin expression (Fig. 3C
; Table 1
). Moreover, methylation was not detected in metastatic OSCC cells that showed E-cadherin expression (Fig. 3C)
. These results indicate that E-cadherin expression and methylation are heterogeneous in metastatic areas.
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and accumulation of ß-catenin protein by Western blot (Fig. 4B)
. E-cadherin protein levels did not change after ZLLL treatment. Because accumulation of p27 after ZLLL treatment was also observed, ZLLL could inhibit proteasome-mediated proteolysis. ß-Catenin protein was not observed in the membrane fraction of MSCC-Inv1 and MSCC-Inv2 cells, but accumulation of membranous ß-catenin was detected after ZLLL treatment (Fig. 4B)
. In Fig. 4A
, the expression level of ß-catenin was very low in the cytoplasm of both parent MSCC-1 cells and MSCC-Inv1 cells. After ZLLL treatment, accumulation of ß-catenin was observed in the cytoplasm of both types of cells. This result indicates that cytoplasmic ß-catenin is constitutionally degraded in both MSCC-1 parent cells and MSCC-Inv1 cells. Therefore, we used whole lysates in the following study (Fig. 4, C and D)
.
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. These findings indicate that membranous ß-catenin may be degraded by ubiquitin-mediated proteolysis in invasive and metastatic OSCC cells. These findings prompted us to test whether ß-Trcp was involved in regulating membranous ß-catenin stability. ß-Trcp1 and ß-Trcp2 are known to be involved in cytoplasmic ß-catenin stability (24)
. To test this, we used the siRNA technique to reduce expression of ß-Trcp1 and ß-Trcp2 in MSCC-1, MSCC-Inv1, and MSCC-Inv2 cells. We used an oligonucleotide that efficiently targets both ß-Trcp1 and ß-Trcp2, as shown previously by Northern blot analysis (24)
. We used IB expression as a control that showed the effect of ß-Trcp siRNA. After ß-Trcp siRNA treatment, phosphorylated IB was observed in all cells, indicating that ß-Trcp was silenced. When compared with these cells transfected with a control double-stranded RNA (dsRNA) oligonucleotide, cells transfected with a dsRNA oligonucleotide corresponding to both ß-Trcp1 and ß-Trcp2 showed no dramatic increase in the levels of ß-catenin (Fig. 4D)
.
Next, to evaluate whether reduced expression of membranous ß-catenin is observed in OSCC cases, we examined the expression of ß-catenin in 10 normal oral mucosa samples, 7 primary OSCCs (tumors were surgically resected from tongue), and 17 metastatic OSCC cases (tumors were surgically resected from the cervical or submandibular lymph node). In all cases of normal oral epithelium, high expression of ß-catenin was observed in the cell membrane (Fig. 4E
; Table 2
). Four of seven cases showed high expression of ß-catenin in the noninvasive area. Interestingly, all cases showed low expression of ß-catenin in the invasive area (Fig. 4E
; Table 2
). Moreover, most of the metastatic OSCC cases (15 of 17 cases) showed reduced expression of membranous ß-catenin (Fig. 4F
; Table 2
). No OSCC cases showed nuclear expression of ß-catenin, and only three metastatic OSCC cases showed cytoplasmic expression of ß-catenin (Table 2)
.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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MSCC-Inv1 and MSCC-Inv2 cells showed reduced expression of E-cadherin and ß-catenin in comparison with parent cells. These highly invasive clones acquired down-regulation of E-cadherin (Fig. 1D)
. This finding is supported by the previous reports (7, 8, 9, 10)
that reduced expression of E-cadherin is well associated with poor differentiation and a higher metastatic potential in OSCC. We also found that adhesion blocking by an anti-E-cadherin antibody increased the invasiveness of the parent MSCC-1 cells, and E-cadherin transfection inhibited the invasion of MSCC-Inv1 cells (Fig. 2)
. We suggest that down-regulation of E-cadherin may be strongly engaged in the invasion of OSCC cells. In fact, down-regulation of E-cadherin in mouse skin carcinoma cells increased motility in vitro and metastatic potential in vivo (30)
. Furthermore, we found that down-regulation of E-cadherin was brought about by CpG methylation around the promoter region in these highly invasive clones (Fig. 3A)
. Interestingly, methylation of E-cadherin was observed in cancer cells of invaded areas and metastatic lesions that showed reduced expression of E-cadherin (Fig. 3, B and C)
. It has been reported that the histological diffuse invasion type of OSCC showed reduced expression of E-cadherin and methylation (31)
. We also found that reduced expression of E-cadherin was heterogeneous within a metastatic OSCCs and that methylation was observed in metastatic cells with reduced expression of E-cadherin (Fig. 3C)
. Chang et al. found that methylation was observed in 67% of nodal metastases of OSCC cases (32)
. From these points of view, we hypothesize that methylation of E-cadherin may be reversible. Therefore, cancer cells with E-cadherin methylation may be focally dissociated at the invading fronts and metastasize to lymph nodes or other organs, and then demethylation may occur in metastatic cancer cells (Fig. 5)
. We emphasize that E-cadherin methylation may play an important role in invasion and metastasis in OSCC progression.
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; Table 2
). We think that reduction of membranous ß-catenin protein may be involved in invasion and metastasis through impaired cell-to-cell adhesion. In our study, nuclear accumulation of ß-catenin was not observed in OSCC cases. Interestingly, the reduced expression of membranous ß-catenin was caused by its protein degradation (Fig. 4, A–C)
. This finding is supported by a recent report (34)
that showed no ß-catenin and AXIN1 gene mutations and no methylation of the CpG island of ß-catenin, AXIN1, and GSK3ß genes in oral cancer cells. Suzui et al. (35)
also demonstrated that no ß-catenin gene mutations were found in rat oral tumors induced by 4-nitroquinoline 1-oxide treatment. Cytoplasmic ß-catenin is known to be phosphorylated by a multimolecule complex consisting GSK3, APC, and AXIN, and then phosphorylated ß-catenin is recognized and ubiquitinated by SCFß-Trcp (15)
. However, degradation of membranous ß-catenin may not be ubiquitinated by SCFß-Trcp because ß-Trcp siRNA did not induce accumulation of ß-catenin in the highly invasive clones (Fig. 4D)
. We suggest that another E3 ubiquitin ligase may be concerned with the degradation of membranous ß-catenin. Recently, it has been reported that during myogenesis, OZZ-E3, a novel SOCS protein, controls the ubiquitination and degradation of the specific pools of ß-catenin located at the sarcolemma (36)
. Because OZZ-E3 is a muscle-specific protein, future studies should be required to clarify the mechanism of membranous ß-catenin degradation in OSCCs and other malignancies. This is the first report regarding the degradation of membranous ß-catenin in epithelial cells, but future studies should be required to clarify the mechanism of the membranous ß-catenin degradation in OSCCs and other malignancies.
In conclusion, our findings in the present study suggest the following: (a) loss of E-cadherin and ß-catenin may be involved in the invasion and metastasis of OSCC cells; (b) OSCC cells with E- cadherin methylation and/or ß-catenin degradation may be able to invade and metastasize (Fig. 5)
; and (c) the method we used for isolation of highly invasive clones may be useful for a study aimed at discovering novel genes involved in invasion and metastasis. We believe that E-cadherin methylation and ß-catenin degradation can be a novel target for inhibition of invasion and metastasis of OSCC as well as a marker for prediction of metastasis.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Requests for reprints: Takashi Takata, Department of Oral Maxillofacial Pathobiology, Division of Frontier Medical Science, Graduate School of Biomedical Sciences, Hiroshima University, 1-2-3 Kasumi, Minami-ku, Hiroshima 734-8553, Japan. Phone: 81-82-257-5634; Fax: 81-82-257-5619. E-mail: ttakata{at}hiroshima-u.ac.jp
Received 2/26/04; revised 4/16/04; accepted 5/14/04.
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