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Lin-7C/VELI3/MALS-3: An Essential Component in Metastasis of Human Squamous Cell Carcinoma

¹ Department of Oral and Maxillofacial Surgery, Tokyo Dental College; Departments of ² Clinical Molecular Biology, ³ Neurological Surgery, and ⁴ Functional Genomics, Graduate School of Medicine, Chiba University; ⁵ Division of Dentistry and Oral-Maxillofacial Surgery, Chiba University Hospital, Chiba, Japan

Requests for reprints: Katsuhiro Uzawa, Department of Clinical Molecular Biology, Graduate School of Medicine, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba 260-8670, Japan. Phone: 81-43-226-2300; Fax: 81-43-226-2151; E-mail: uzawak{at}faculty.chiba-u.jp.

	Abstract

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Using proteomic selection, functional verification, and clinical validation, we identified specific down-regulation of Lin-7C/VELI3/MALS-3 (Lin-7C), which marks oral squamous cell carcinoma (OSCC) metastasis. Despite a rarity of sequence variations in the Lin-7C gene in both primary OSCC and OSCC-derived cells, a high prevalence of hypermethylation was detected in the CpG island region that strongly correlated with its down-regulation. Inducible Lin-7C mRNA by experimental demethylation was found in all OSCC cells tested. Overexpression of the Lin-7C gene in an OSCC cell clone does not contribute to underproliferation but results in a noninvasive phenotype with elevated ß-catenin expression. Experimental metastases in multiple organs of immunodeficient mice were inhibited in cells expressing Lin-7C. Finally, the Lin-7C expression status in primary tumors afforded significantly (P < 0.001) high accuracy for predicting lymph node metastasis. These results establish Lin-7C as a novel target of early detection, prevention, and therapy for OSCC metastasis. [Cancer Res 2007;67(20):9643–8]

	Introduction

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Squamous cell carcinoma (SCC), which is by far the most common malignant neoplasm of the oral cavity, is one of the leading causes of death from cancer (1). The vast majority of patients who have either suspected or proven metastases in regional lymph nodes are candidates for composite resection in which the lesion, surrounding tissues, and lymph nodes of the neck are all removed.

The carcinogenesis of oral SCC (OSCC) is thought to be a complex, multistep phenomenon in which a variety of genetic alterations can be segregated into early to late stages. During this process, loss of metastasis suppressor gene(s) function should be a crucial step in the progression of cancer cells from a nonmetastatic to a metastatic phenotype. Recent studies have identified the correlation between gene expression patterns and lymph node metastasis in OSCC (2, 3). Yet, there is no critical determinant that can be used to identify which primary lesions will develop into metastatic OSCC.

It has recently been recognized that proteome analysis could be a powerful analytic technology developed to enhance our study of the diagnosis, prognosis, treatment, and prevention of human diseases (4–6). In this context, two-dimensional PAGE is the principal tool in proteomics that can resolve thousands of proteins in one experiment and provide the highest resolution in protein separation. Moreover, fluorescent two-dimensional differential in-gel electrophoresis (2D-DIGE) allows the multiplex analysis of two sample proteomes on the same gel (7, 8). In a recent study, we used a model system of cell lines derived from primary normal oral tissue and oral cancer–derived cells to investigate the difference of protein expression patterns by using the 2D-DIGE combined with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS; ref. 9). We identified groups of proteins of which the expression differed between normal oral keratinocytes (NOK) and OSCC-derived cells. We further validated that Lin-7C/VELI3/MALS-3 (Lin-7C) mediates OSCC metastasis with ß-catenin signaling and is clinically linked to the development of cervical lymph node metastasis when the Lin-7C expression level decreases in primary tumors.

	Materials and Methods

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Cells. All the human OSCC-derived cell lines (Ca9-22, Ho-1-N-1, Ho-1-u-1, HSC-2, HSC-3, HSC-4, OK92, Sa3, and H1) and primary cultured NOKs used as a normal control were cultured as described (10).

Tissue samples and nucleic acid isolation. Tumors with patient-matched normal oral tissues were obtained at the time of surgical resection at Chiba University Hospital after informed consent was obtained from the patients according to a protocol that was reviewed and approved by the institutional review board of Chiba University. Histopathologic diagnosis of each cancerous tissue was done according to the International Histological Classification of Tumours. Genomic DNA was extracted by a proteinase K digestion procedure and total RNA was prepared using Trizol reagent (Invitrogen) according to the manufacturer's protocol.

Proteomic analyses. We did proteomic profiling of NOKs and OSCC-derived cell lines (HSC-2 and HSC-3) using fluorescent 2D-DIGE and MALDI-TOF-MS fingerprinting in triplicate as described previously (9). Each protein extract (50 µg) was used for the 2D-DIGE analysis.

Pathway analysis. The target gene lists were overlaid on a cellular pathway map in the Ingenuity Pathway Analysis (IPA) software (Ingenuity Systems; ref. 11). The expression levels of the mRNA of target genes were examined using quantitative reverse transcription-PCR analyses. The primer sequences of the genes related to Lin-7C are shown in the Supplementary Data.

Immunohistochemical staining. Immunohistochemical staining was done on 4-µm paraffin-embedded specimens as described with anti-Lin-7C (Santa Cruz Biotechnology) at a dilution of 1:500. All slides were finally lightly counterstained with hematoxylin and mounted. To quantitate Lin-7C protein expression, a scoring method was used as described previously (10). Cases with a Lin-7C immunohistochemical score of <59 (minimum score of normal tissues) were considered negative.

mRNA expression analyses. The expression levels of Lin-7C mRNA were examined by quantitative reverse transcription-PCR with a single method using a LightCycler FastStart DNA Master SYBR Green kit (Roche) according to the manufacturer's instructions. The primer sequences used for analysis of Lin-7C mRNA expression were shown in the Supplementary Data. The sequence of specific primers was checked before use to avoid amplification of genomic DNA or pseudogenes by the Primer3 program.⁶

Mutational analyses. To identify sequence variations, the Lin-7C gene was analyzed based on a PCR direct sequencing method as described previously (10). Five sets of oligonucleotide primers summarized in the Supplementary Data were used to amplify the entire coding region (exons 1–5) of the Lin-7C gene.

Methylation analyses. We analyzed the methylation status of the CpG island region of the Lin-7C gene using a PCR-based methylation assay. The DNA samples were digested with mCpG-sensitive BstUI (1 µg/unit; New England Biolabs) at 60°C for 16 h. The BstUI-digested DNAs were then amplified with specific primers shown in the Supplementary Data. Peripheral blood DNA treated in vitro with SssI methylase (New England Biolabs) was used as a positive control, and the SssI-untreated DNA was used as a negative control. To assess reactivation of Lin-7C mRNA expression, a demethylating assay was done as described (12).

Functional analyses of Lin-7C. Human Lin-7C cDNA was cloned into a pME18SFL3 expression vector for transient transfection experiments (13). The nine OSCC-derived cell lines were transfected with pME18SFL3 encoding Lin-7C cDNA using the nonliposomal formulation FuGENE6 transfection reagent (Roche). All experiments using these cells were done 48 h after transfection. Mock transfection of the nine OSCC-derived cell line cultures with pME18SFL3 expression vector alone served as a control. Quantitative reverse transcription-PCR analyses and immunofluorescence were done to confirm transfection efficiency (13). To determine the motility capacity of human OSCC cells, a wound-healing assay was done as described previously with some modifications (14). To evaluate the invasive capacity of both transfected and mock cells, invasion assays were done with the QCM 96-well Cell Invasion Assay kit (Chemicon; ref. 13).

In vivo experimental metastasis assays. A full-length human Lin-7C construct was purchased from Toyobo and cloned into pcDNA4/TO vectors (Invitrogen). The vector containing Lin-7C of the empty pcDNA4/TO vector was transfected into Ca9-22 cells with the FuGENE6 kit according to the manufacturer's protocol. Stable expression clones were selected with zeocin (Sigma) for and screened by Western blotting using monoclonal antibody for Lin-7C (Santa Cruz Biotechnology). Cells (2 x 10⁵) from each clone were injected directly into the tongues of the BALB/cAnNcrj-nu/nu mice (Charles River Japan), which were sacrificed after 6 weeks. We then excised several organs, including the tongue, submandibular glands, lungs, and liver, and DNA was extracted from them for PCR amplification of the human-specific Alu sequence (15). All mice were maintained under specific pathogen-free conditions. All animal experiments were conducted in accordance with the guidelines of the Chiba University laboratory animal center.

	Results

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Proteomic analyses. An average of 2,500 protein spots was detected across all five 2D-DIGE gels (Fig. 1A ). A 2-fold or greater change in protein expression was considered significant. In OSCC-derived cell lines (HSC-2 and HSC-3), we found 226 differentially expressed protein spots compared with the NOKs. Among them, 92 were up-regulated and 134 were down-regulated. Among the protein spots detected, 9 proteins were commonly up-regulated and 15 were commonly down-regulated in both cell lines studied (Supplementary Table S1). MALDI-TOF-MS measurement and database matching identified most of the matched proteins with high sequence coverage and mass accuracy (Fig. 1B–D).

View larger version (20K): [in this window] [in a new window] [Download PPT slide]   Figure 1. Comparative analysis of OSCC-derived cells and NOKs using the 2D-DIGE system. A, typical 2D-DIGE gel images of OSCC-derived cells (HSC-2) and NOKs. The protein lysates are labeled with either Cy3 (green, NOKs) or Cy5 (red, HSC-2) and subjected to 2D-DIGE using Immobiline Dry IPG strips (pH 3–10). IEF, isoelectric focusing. B, close-ups of 2D-DIGE gel images show significant (P < 0.001) down-regulation in the HSC-2 cell lines compared with the NOKs. C, three-dimensional simulation of up-regulated and down-regulated protein spots. D, top, results of MALDI-TOF-MS analysis of the spot obtained from NOKs; bottom, amino acid sequences analyzed for Lin-7C by peptide mass fingerprinting analysis. Letters in red, protein sequences characterized by MALDI-TOF-MS analysis.

View larger version (48K): [in this window] [in a new window] [Download PPT slide]   Figure 3. Functional analyses of Lin-7C by IPA. A, characterization of the genetic networks that could affect Lin-7C gene expression using IPA. When examined by quantitative reverse transcription-PCR analyses, the mRNA expression levels of these 20 genes are up-regulated or down-regulated in Lin-7C–transfected OSCC-derived cell lines compared with mock-transfected OSCC-derived cell lines and untreated cell lines. On Western blotting analyses (B) and immunofluorescence (C), no significant differences in the CTNNB1 (ß-catenin) and CASK (Lin-2) expression patterns are seen between the mock-transfected OSCC-derived cell lines (lane –) and the untreated cell lines (lane N). In contrast, all Lin-7C–transfected OSCC-derived cell lines (lane +) have a strong immunoreaction for ß-catenin and Lin-2.

View larger version (64K): [in this window] [in a new window] [Download PPT slide]   Figure 2. Gene expression profiling of Lin-7C. A, immunohistochemical staining of Lin-7C in normal and cancerous oral tissues. Top, typical examples of Lin-7C–positive staining; bottom, examples of the negative cases. Normal oral tissues show strong Lin-7C protein expression limited to the cell membrane. A typical example of primary OSCC without a metastatic lesion is positive for Lin-7C. In contrast, primary cases with a metastatic lesion and the metastatic OSCC itself are negative for Lin-7C. Original magnification, x400. B, state of Lin-7C protein expression in primary OSCCs with node-positive (pN+) or node-negative (pN–) metastatic lesions. The Lin-7C immunohistochemical (IHC) scores were calculated as follows: immunohistochemical scores = the percentage of positive tumor cells x staining intensity. Both the test set (n = 80) and the validation set (n = 30) indicated that node-positive cases have significantly (P < 0.001) decreased Lin-7C protein expression. C, bottom, typical results of direct DNA sequencing of PCR products from a primary case of OSCC (case 20) show one-base nucleotide substitution in exon 1 (codon 6) of the gene compared with the DNA from normal tissues. D, typical results of the methylation assay for OSCC-derived cells and OSCC cases. Lin-7C methylation is seen in all OSCC cell lines (top), which show reduced Lin-7C mRNA expression compared with the NOKs. Bottom, in addition, whereas no normal tissue has methylated Lin-7C, most primary tumors do. DNA from NOK treated in vitro with SssI methylase is used as a positive control (lane PC) for methylated alleles. SssI-untreated DNA is used as a negative control (lane NC) for methylated genes. M, molecular marker.

Functional analyses of Lin-7C gene in OSCC cells. Although two clones from the Ca9-22 cells (Lin7 a-1 and Lin7 5-5) that stably expressed the Lin-7C gene showed no significant differences in their phenotypic changes and the proliferation rate, they showed significant inhibition of motility and invasiveness as determined by a wound-healing assay and invasive assay when compared with the cells with empty vector controls (mock experiment, Fig. 4A–C). To determine whether down-regulation of Lin-7C induces metastasis in vivo, we then injected the Lin-7C–expressing cells (Lin7 a-1 and Lin7 5-5) into the tongue of nude mice. Although no visible metastatic lesions were observed in distant organs, such as the lung, liver, kidney, and colon, the human-specific Alu sequence was detected only in the organs of the mice injected with the Ca9-22 cells with empty vector controls (Fig. 4D).

View larger version (43K): [in this window] [in a new window] [Download PPT slide]   Figure 4. In vivo experimental metastasis assays. A, human full-length Lin-7C cDNA was cloned into an expression vector for stable transfection experiments. Western blot analysis was done to confirm transfection efficiency. No significant difference in the Lin-7C expression pattern is seen between mock-transfected Ca9-22 cell lines (MOCK) and untreated cell lines. In contrast, the Lin-7C–transfected OSCC-derived cell lines (Lin7 5-5 and Lin7 a-1) had a strong immunoreaction for Lin-7C. B, enhanced invasion by cells expressing Lin-7C in wound-healing assays. After uniform wounds were made in confluent cultures of different Lin-7C–expressing and mock-transfected cells, the extent of closure was visually monitored. Eight hours later, the wound is completely sealed in the cultures of Ca9-22 mock-transfected cells, whereas there is still a gap in the Lin7 5-5 and Lin7 a-1 cells. C, the invasive activity of the Lin-7C–transfected cell lines (Lin7 5-5 and Lin7 a-1) and the mock-transfected cell lines was tested using the ECMatrix invasion chamber assay. Transfection of OSCC-derived cell lines with Lin-7C produces a significant (P = 0.02) decrease in invasive activity. D, representative results of an in vivo metastasis assay in five mice. Whereas mock-transfected Ca9-22 cells spontaneously metastasize into the submandibular glands, lungs, and liver, Lin-7C–expressing Ca9-22 cells (Lin7 a-1) are not detected in these organs, suggesting that Lin-7C inhibits micrometastasis of Ca9-22 cells.

	Discussion

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Although Lin-7C is on 11p14, a region that is deleted in 38% of human head and neck cancers (16), the identity of crucial gene(s) for oral cancer, where Lin-7C is located, is still unclear. Lin-7C contains one PDZ domain that is known to mediate protein-protein interactions with COOH-terminal tails of transmembrane protein. Recent studies have reported that although the Lin-7C PDZ protein is a component of the mature cadherin-based junctional domain in glioma cells, the immature junctions of migrating cells fail to accumulate Lin-7C, suggesting that its presence could be considered a marker of junctional maturity and invasiveness. An interesting study has shown that microtubule disruption of the cadherin-catenin complex resulted in relocalization of the E-cadherin-catenin-actin complex in normal human epidermal keratinocytes but not in squamous cancer cell lines (17). Thus, the loss of cell-cell adhesion via the cadherin-catenin complex in SCC cells is likely due to this irreversible modification. Lin-2 is a cell adhesion molecule and also has a PDZ domain. The central PDZ domain of Lin-2 binds to the cytoplasmic tails of several cell surface proteins. However, no relationship between human carcinogenesis and Lin-2 status has been confirmed thus far. No reports have been published about an association between Lin-2 and human carcinogenesis, and further studies should evaluate whether down-regulation of Lin-2 may play a role in oral tumorigenesis. ß-catenin, whose function is cellular adhesion through the Wnt signaling pathway (18), is down-regulated in the metastatic lesions of OSCC (19), with decreased expression of E-cadherin that is associated with specific stages of tumor differentiation (20). Reduced expression of ß-catenin has been proved to be due to degradation through the ubiquitin-proteasome pathway or due to structural abnormalities of the gene. In the current study, overexpression of Lin-7C results in repression of Lin-2 and ß-catenin and reduction of invasive/metastatic phenotypes in OSCC cells in vitro (Fig. 3B and C) and in vivo (Fig. 4A–D ), suggesting that Lin-7C expression in OSCC is part of the ß-catenin signaling pathway that regulates ß-catenin and that this pathway may be one of the most important factors in metastatic suppression.

Whereas inactivation of tumor suppressor genes caused by deletion or mutation enhances tumor growth, the loss of Lin-7C function in OSCC induced tumor cell invasion without affecting the growth of the primary tumor. Therefore, we have hypothesized that Lin-7C might suppress metastasis in oral carcinogenesis and that Lin-7C up-regulation, possibly by demethylation (Supplementary Fig. S2), in primary OSCC and probably other types of human cancers may suppress tumor invasion and metastasis. The crucial role of the significant down-regulation of Lin-7C could determine the type of primary tumor that arises in metastatic phenotype. It will be important to design clinical studies to assess the predictive value of Lin-7C down-regulation in patients with OSCC because our test set consisting of newly diagnosed OSCC (n = 30) has been divided into two groups (i.e., those with and without regional metastasis) based on the status of Lin-7C expression in primary tumors.

	Acknowledgments

 
Grant support: The 21st Century Center of Excellence Programs Grant from the Ministry of Education, Culture, Sports, Science and Technology of Japan (H. Tanzawa).

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.

We thank Dr. Takaki Hiwasa (Department of Biochemistry and Genetics, Chiba University Graduate School of Medicine) for invaluable advice in proteomic analysis and Lynda C. Charters for editing this manuscript.

	Footnotes

 
Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/).

T. Onda and K. Uzawa contributed equally to this work.

⁶ http://www-genome.wi.mit.edu/cgibin/primer/primer3_www.cgi

Received 5/25/07. Revised 8/ 3/07. Accepted 8/30/07.

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