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RESEARCH REPORT |
1 Department of Oral Surgery, Graduate School of Dentistry, and
2 Department of Biochemistry, School of Dentistry at Tokyo, The Nippon Dental University, 1-9-20 Fujimi, Chiyoda-ku, Tokyo 102-8159, Japan;
* corresponding author, KIMAI{at}Tokyo.ndu.ac.jp
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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KEY WORDS: ß-catenin • EMT • invasion • oral carcinoma • WNT
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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The WNT family has a crucial role in development and constitutes 19 members in the human genome. WNT1 was identified as an oncogenic gene activated by chromosomal integration of Mouse Mammary Tumor Virus. Ligation of WNT to cell-surface receptors, FRIZZLED (FZ) and LDL receptor-related protein, sparks signaling pathways mediated by ß-catenin (CTNNB1) or others (Seidensticker and Behrens, 2000). CTNNB1-mediated signaling has been characterized extensively (Seidensticker and Behrens, 2000). In this pathway, FZ abrogates kinase activity of glycogen synthase kinase 3-ß (GSK3B), liberating CTNNB1 from degradation and increasing the cytoplasmic-free CTNNB1 pool. An excess amount of CTNNB1 translocates into the nucleus and transcribes target genes. In the absence of WNT, GSK3B forces CTNNB1 to degrade, resulting in a decrease of the free CTNNB1 pool (Seidensticker and Behrens, 2000; Lo Muzio, 2001).
WNT signaling initiates proliferation, dedifferentiation, and EMTs in various types of carcinoma cells (Eger et al., 2000; Lo Muzio, 2001). Recent evidence increases a panel of WNT target genes, such as c-MYC and extracellular matrix (ECM)-degrading endoproteinases (www.stanford.edu/~rnusse/wntwindow.html). c-MYC is involved in tumorigenesis and cell proliferation (Lutz et al., 2002). Degradation of the ECM is an indispensable step in tumor invasion and metastasis, and matrix metalloproteinases (MMPs) have a central role in such degradation (Imai et al., 1995a, 1997a; Ohuchi et al., 1997). Recent studies emphasize a role for membrane-type 1 MMP (MT1-MMP) in cancer cell invasion and progression of other diseases (Imai et al., 1996, 1997b; Ueno et al., 1997). It is known that WNT signaling directly up-regulates MT1-MMP expression (Takahashi et al., 2002). Therefore, activation of the WNT signaling pathway can have a significant impact on tumor progression. However, no examination has been made of WNT expression in oral carcinomas (Lo Muzio, 2001). In the present study, we developed an expression panel of WNT family members and demonstrated activation of WNT signaling in carcinoma cells. Immunohistochemical staining indicated that the signaling was prominent at the invasive front in the carcinoma tissues.
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MATERIALS & METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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Reverse-transcription/PCR
Total RNA was isolated with the use of TRIzol reagent (Invitrogen) followed by RNase-free DNase I treatment. After reverse transcription (RT) with SuperScript II and random hexamer (Invitrogen), we performed PCR amplification by running 30 cycles under the following conditions: 94°C for 40 sec, 54°C for 40 sec, and 72°C for 1 min. Gene-specific primer sets for WNTs or GAPDH are listed in the Table
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Immunocytochemistry
Carcinoma cell lines (TSU, HOC313, HSC2, and SCCKN) were cultured on glass slides (Lab-Tek Chamber II, NUNC, Naperville, IL, USA) and fixed with 1% paraformaldehyde. The cells were reacted with primary antibodies to WNT3 (10 µg/mL), CTNNB1 (4 µg/mL), or GSK3B (4 µg/mL, Cell Signaling Technology, Beverly, MA, USA) for 16 hrs at 4°C. Alexa Fluor 546 anti-rabbit or -goat IgG or Alexa Fluor 488 anti-mouse IgG antibodies (Molecular Probes, Eugene, OR, USA) were used for secondary antibody.
Immunoblotting
Nuclear extracts of TSU, HOC313, HSC2, or SCCKN cells were prepared by the method described elsewhere (Lee et al., 1988). Total cell lysates (35 µg) or nuclear extracts (20 µg) were size-fractionated by SDS-PAGE and electrotransferred onto nitrocellulose membranes. The membrane was probed with anti-CTNNB1, -GSK3B, or -ß-actin (Sigma-Aldrich) antibodies and then with biotinylated secondary antibodies, and the color developed with avidin-biotin-complex and 3,3'-diaminobenzidine tetrahydrochloride.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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). Among 19 members of the family, WNT3, 3a, 4, 5a, 5b, 6, 7a, 7b, 10b, 11, and 14 were amplified in all of the carcinoma cell lines, except for HSC2 cells. Normal keratinocytes expressed WNT5a, 5b, 6, and 7a, and GF12 gingival fibroblasts amplified WNT2, 2b, 3, 5a, 5b, 11, 14, and 16 (Fig. 1B). Keratinocyte-type WNTs (6 and 7a) were most commonly expressed, and fibroblast-type WNTs (3, 11, and 14) were less frequently expressed. Other cell-type WNTs (3a, 4, 7b, and 10b) were also ectopically identified in the carcinoma cell lines examined. Specific amplification of each WNT gene was clarified by sequence analysis (data not shown).
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). Immunostaining and Western blot analyses showed weak reduction of GSK3B in HSC2 cells (Figs. 2A, 2B). However, immunostaining of CTNNB1 in WNT-expressing HOC313, TSU, and SCCKN cells exhibited accumulation in the nucleus and diffuse cytoplasmic distribution (Fig. 2A). In contrast, HSC2 cells localized CTNNB1 at the cell-cell boundaries. Since CTNNB1 is crucial for the formation of E-cadherin-mediated cell-cell adhesion (Nagafuchi et al., 1994), it is bifunctional, undertaking WNT signaling and cell-cell adhesion (Gottardi et al., 2001). Western blot analysis also showed that increased amounts of the free CTNNB1 pool were in the WNT-expressing cells compared with HSC2 cells (Fig. 2B). Nuclear extracts of HOC313, TSU, or SCCKN cells reacted to CTNNB1 antibody, whereas no reaction was observed in the HSC2 cells.
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). Western blotting analyses also showed selective expression of MT1-MMP in the WNT-expressing TSU, HOC313, and SCCKN cells (Fig. 2C).
Immunostaining of WNT3 and CTNNB1 in Oral Carcinoma Tissues
Immunohistochemistry was performed on 42 carcinoma tissue specimens (Fig. 3). WNT3 staining was observed in carcinoma cells in 28 cases (57%), predominantly at the invasive front. Endothelial cells, fibroblast-like cells, and macrophage-like cells were all positively stained. No staining was observed when anti-WNT3 antibody was substituted for non-immune goat IgG negative controls (data not shown). Normal oral tissues did not stain with the anti-WNT3 antibody. CTNNB1 primarily stained the cell-cell boundaries of the carcinoma cells. However, in the cells at the invasive front, CTNNB1 was localized to the nucleus and diffusively to cytoplasm. Cytoplasmic staining was evident in endothelial cells, fibroblast-like cells, and macrophage-like cells (data not shown). Normal epithelial cells stained only at the cell-cell boundaries of the suprabasal cell layer (data not shown).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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WNT3 is a typical and powerful member of the family which activates CTNNB1-mediated signaling (Seidensticker and Behrens, 2000; Lo Muzio, 2001). Therefore, we examined the expression pattern of WNT3 and activation of the CTNNB1 signaling in tissue sections of oral carcinomas. WNT3 immunostaining demonstrated ectopic expression in carcinoma cells at the invasive front. CTNNB1 also localized in the nuclei of carcinoma cells at the invasive front, where cells evade cell-cell adhesion and gain the characteristics of EMTs (Imai et al., 1995b; Brabletz et al., 2001). Analogous findings have been reported in colorectal carcinomas (Kirchner and Brabletz, 2000; Brabletz et al., 2001; Takahashi et al., 2002). WNT signaling triggers EMTs, and nuclear localization of CTNNB1 correlates with the induction of EMTs (Eger et al., 2000). EMTs are particularly prominent at the invasive front and predispose carcinomas to a more advanced state of progression (Birchmeier et al., 1996). The fibroblast-type WNTs and CTNNB1-mediated signaling are documented to initiate malignant transformation and enhance cellular proliferation, dedifferentiation, and invasion and metastasis (Lo Muzio, 2001). Therefore, enhanced expression and signaling of the fibroblast-type WNT have a key role in the induction of EMTs and tumor progression.
Although we could not exclude the possibility that WNT directly initiated migration of carcinoma cells, it seems likely that WNT signaling increases local invasiveness of carcinoma cells through up-regulation of ECM-degrading endoproteinases. In fact, only WNT-expressing carcinoma cells synthesized MT1-MMP. It is known that MT1-MMP is up-regulated in metastatic oral carcinomas (Shimada et al., 2000). In colorectal carcinomas, MT1-MMP is a direct target gene of WNT signaling, and MT1-MMP and nuclear CTNNB1 immunolocalize in carcinoma cells in almost an identical pattern (Takahashi et al., 2002). Upstream sequences during induction of MMPs and MT1-MMP in vivo are not clearly defined. However, these observations are highly indicative of a key role of WNT signaling in the induction of MT1-MMP, contributing to tumor invasion.
Expression of c-MYC is regulated by transcriptional and/or signal transduction hierarchies (Lutz et al., 2002). Recently, van de Wetering et al.(2002) demonstrated that WNT signaling initiates proliferation and suppresses differentiation of colorectal carcinoma cells through induction of c-MYC, which negatively regulates the cell-cycle inhibitor p21CIP1/WAF1. Although c-MYC protein accumulated in the nucleus of HOC313 and TSU cell lines, SCCKN cells, which also activated WNT signaling, did not express c-MYC. There might be WNT-independent and/or negative regulatory pathway(s) in the regulation of c-MYC expression in oral carcinoma cells.
WNT3 immunohistochemistry stained endothelial cells, fibroblast-like cells and macrophage-like cells. Activation of the WNT signaling pathway stimulates proliferation of endothelial cells (Wright et al., 1999). WNT expression in endothelial cells might promote angiogenesis in oral carcinoma tissues. Immunostaining of WNT3 in fibroblast-like cells also suggests a role in the desmoplastic response in carcinoma tissues, since it has been reported that WNT up-regulates proliferation and collagen synthesis in fibroblasts (Young et al., 1998; Surendran et al., 2002). Although the biological role of WNT expression in macrophages is not clear, a similar finding was reported in human colorectal carcinomas (Smith et al., 1999). Therefore, the WNT family would mediate cross-talking between carcinoma cells and stromal cells. Further investigation will clarify a role for WNT-mediated cross-talking in the pathology of oral carcinomas.
The HSC2 cell line did not express any members of the WNT family. This suggests the presence of WNT-independent pathways in malignant transformation and/or the maintenance of malignant phenotypes. However, mutations of the WNT signaling molecules make constitutive activation of the signaling without expression of the WNT molecule (Morin et al., 1997; Rubinfeld et al., 1997; Satoh et al., 2000). Although these mutations should accumulate CTNNB1 in the cytoplasm and nucleus, we could not exclude the possibility that the mutations may exceed a signaling threshold with an undetectable level of CTNNB1 protein in HSC2 cells. Future study should disclose genetic aberrations in oral carcinoma cells.
The present study demonstrated that squamous carcinoma cells of the oral cavity express a set of WNT genes and activate signaling, and suggests that the signaling is predominantly activated in carcinoma cells at the invasive front, wherein carcinoma cells accumulate EMTs. The activation state of WNT signaling correlates with MT1-MMP expression, which accelerates invasion into territorial matrix. Analysis of the data in our study suggests the possibility that inhibition of WNT signaling could be a potential target for suppression of tumor progression.
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ACKNOWLEDGMENTS |
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Received May 7, 2003; Last revision January 14, 2004; Accepted January 16, 2004
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REFERENCES |
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