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Activation of WNT Family Expression and Signaling in Squamous Cell Carcinomas of the Oral Cavity
1 Department of Oral Surgery, Graduate School of Dentistry, and Correspondence: * corresponding author, KIMAI{at}Tokyo.ndu.ac.jp
The WNT family activates an oncogenic signaling mediated through β-catenin and is up-regulated in a variety of malignant neoplasms. The signaling translocates β-catenin into the nucleus and stimulates carcinoma cells in the epithelial-mesenchymal transition (EMT). However, WNT expression and signaling in oral carcinomas have not been examined. The present study focused on unveiling the involvement of WNTs in oral carcinomas, and showed that carcinoma cells express 11 of 19 WNT family members by reverse-transcription/PCR. WNT-expressing carcinoma cells exhibited increased β-catenin levels in the cytoplasmic pool and translocation to the nucleus. The activation state of signaling correlated with the expression of membrane-type 1 matrix metalloproteinase, which degrades territorial matrices in carcinoma invasion. Immunohistochemistry disclosed that WNT3 expression and nuclear localization of β-catenin were predominant in carcinoma cells at the invasive front. These results suggest that enhanced WNT expression and signaling accelerate the progression of carcinomas via activating EMTs and local invasiveness.
Key Words: β-catenin • EMT • invasion • oral carcinoma • WNT
Oral squamous cell carcinoma is the most common neoplasm of the head and neck. Carcinoma cells accumulate a series of genetic and/or epigenetic changes and altered phenotypes during tumor progression. Loss of the epithelial morphology and acquisition of mesenchymal characteristics, epithelial-mesenchymal transition (EMT), are typical for carcinoma cells during tumor progression and correlate with local invasiveness and metastatic potential of the tumor. Such changes will associate with aberrant expression of developmentally regulated and mesenchymal cell-related genes (Birchmeier et al., 1996; Mizunuma et al., 2003). Studying the gene expression involved in EMTs may provide insights into the mechanism of tumor progression. 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 β-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-β (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.
Cell Lines and Tissue Samples Oral squamous carcinoma cell lines (Ca9.22, Ho1u1, HOC313, HSC2, HSC3, KOSC2, KOSC3, OSC19, SCCKN, SCCTF, and TSU) were obtained from the Cell Resource Center (Sendai, Japan), the Health Science Research Resources Bank (Osaka, Japan), or the RIKEN Cell Bank (Tsukuba, Japan). Normal gingival fibroblasts (GF12) (Takahashi et al., 1997) were used as a control. Normal gingival keratinocytes from patients undergoing dental surgery at Nippon Dental University Hospital were obtained under an informed consent protocol that was reviewed and approved by the Institutional Review Board, and were primary-cultured in defined keratinocyte-SFM (Invitrogen, Grand Island, NY, USA).
Reverse-transcription/PCR
Tissue Specimen Selection and Immunohistochemistry Incisional or excisional biopsy specimens from 42 patients with oral squamous cell carcinomas were collected from the files of Kanazawa University Hospital. Clinical and pathological data were obtained from the patients’ medical records and The Kanazawa University Hospital Surgical Pathology Files. These samples were immediately fixed in 10% neutral buffered-formalin and embedded in paraffin wax. Unstained tissue sections (4 µm) were incubated with goat anti-WNT3 (10 µg/mL, Santa Cruz Biotechnology, Santa Cruz, CA, USA) or mouse anti-CTNNB1 (5 µg/mL, BD Bioscience, San Diego, CA, USA) antibodies for 12 hrs at 4°C. Biotinylated anti-goat or -mouse IgG antibodies (DAKO, Glostrup, Denmark) were used for secondary antibody, followed by incubation with avidin-biotin complexes (Vector Laboratories, Burlingame, CA, USA). The color was developed with 3,3'-diaminobenzidine tetrahydrochloride (Sigma-Aldrich, St. Louis, MO, USA) and counterstained with hematoxylin.
Immunocytochemistry
Immunoblotting
Expression of the WNT Family in Oral Carcinomas We performed RT-PCR for WNT family members and generated a panel of the expression pattern (Fig. 1A  ). 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).
Activation of WNT Signaling Pathway We examined protein expression by immunohistochemistry and Western blot. WNT3-specific antibody stained HOC313 and TSU cells, but not SCCKN and HSC2 cells (Fig. 2A ). 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.
Activation of Downstream Targets of WNT Signaling Analysis of our data above demonstrates the activation of the WNT signaling pathway in oral carcinoma cells. Among WNT target genes, c-MYC oncogene and MMPs have been well-documented to accelerate tumor progression (Ueno et al., 1997; Shimada et al., 2000; Lutz et al., 2002). Therefore, we examined the expression of c-MYC and MT1-MMP in the cell lines expressing WNT genes. Accumulation of c-MYC in the nucleus was evident in TSU and HOC313 cells but not in HSC2 cells. Unexpectedly, c-MYC was negligible in SCCKN cells (Fig. 2C  ). 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
Several studies emphasize that aberrant activation of the WNT signaling pathway contributes to neoplastic transformation and EMTs toward progressive tumors. The present study is the first demonstration that oral squamous cell carcinomas express WNT members and activate the signaling pathway. Profiling WNT genes by RT-PCR indicates that oral carcinoma cells express a set of WNT genes and suggests that a mixed population of WNT family members may affect the biological state of carcinoma cells. Identification of WNT members in normal gingival keratinocytes demonstrates that oral carcinoma cells most frequently expressed keratinocyte-type WNTs. In addition, carcinoma cells also miss-express sets of fibroblast-type WNTs, indicating that oral carcinoma cells of epithelial origin ectopically activate fibroblast-type WNT expression, including WNT3. Ligation of the fibroblast-type WNTs to the cell-surface receptor transmits a signal mediated through CTNNB1. This signal is one of the most characterized among the WNT-dependent signaling pathways (Seidensticker and Behrens, 2000; Lo Muzio, 2001). Immunocytochemistry and Western blot showed that WNT-expressing carcinoma cells increased a pool of cytoplasmic-free CTNNB1 and accumulation in the nucleus, but not WNT-negative cells. Analysis of these data clearly illustrates activation of the CTNNB1-mediated WNT signaling in oral carcinoma cells. 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.
We are grateful to Drs. Sato (Nippon Dental University), Yamamoto, and Kawashiri (Kanazawa University) for the generous gift of GF12 gingival fibroblasts and tissue specimens, respectively. We also thank Dr. D’Armiento (Columbia University) for critical reading of the manuscript. This study was supported by a grant from the Uehara Memorial Foundation to K. Imai. Received for publication May 7, 2003. Revision received January 14, 2004. Accepted for publication January 16, 2004.
Journal of Dental Research, Vol. 83, No. 4,
327-332 (2004) This article has been cited by other articles:
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