The existence of tumor-suppressor genes was originally demonstrated by functional complementation through whole-cell and microcell fusion. Transfer of chromosome 11 into a human non-small-cell lung cancer (NSCLC) cell line, A549, suppresses tumorigenicity. Loss of heterozygosity (LOH) on the long arm of chromosome 11 has been reported in NSCLC and other cancers. Several independent studies indicate that multiple tumor-suppressor genes are found in this region, including the gene PPP2R1B at 11q23-24 (ref. 7). Linkage studies of NSCLC are precluded because no hereditary forms are known. We previously identified a region of 700 kb on 11q23.2 that completely suppresses tumorigenicity of A549 human NSCLC cells. Most of this tumor-suppressor activity localizes to a 100-kb segment by functional complementation. Here we report that this region contains a single confirmed gene, TSLC1, whose expression is reduced or absent in A549 and several other NSCLC, hepatocellular carcinoma (HCC) and pancreatic cancer (PaC) cell lines. TSLC1 expression or suppression is correlated with promoter methylation state in these cell lines. Restoration of TSLC1 expression to normal or higher levels suppresses tumor formation by A549 cells in nude mice. Only 2 inactivating mutations of TSLC1 were discovered in 161 tumors and tumor cell lines, both among the 20 primary tumors with LOH for 11q23.2. Promoter methylation was observed in 15 of the other 18 primary NSCLC, HCC and PaC tumors with LOH for 11q23.2. Thus, attenuation of TSLC1 expression occurred in 85% of primary tumors with LOH. Hypermethylation of the TSLC1 promoter would seem to represent the 'second hit' in NSCLC with LOH.

We have recently identified a tumor suppressor gene TSLC1 on chromosome 11q23.2 by functional complementation of a human lung cancer cell line, A549, through suppression of tumorigenicity in nude mice (1). Furthermore, we have demonstrated the two-hit inactivation of TSLC1 in primary non-small cell lung cancer, hepatocellular carcinoma, and pancreatic cancer, implying its involvement in various human cancers (1). TSLC1 encodes a member of the immunoglobulin superfamily proteins comprising three Ig-like C2-type domains, a single hydrophobic membrane-spanning ␣-helix, and a cytoplasmic domain containing a putative signaling motif (1). From the significant homology of its extracellular domain with those of NCAM1 and NCAM2, 1 we have inferred that TSLC1 is capable of mediating cell-cell interaction.Cell adhesion molecules generally fall into four major classes: the cadherins, the integrins, the selectins, and the Ig superfamily. Among them, Ig superfamily cell adhesion molecules (IgCAMs) are the largest, numbering well over 100 members in vertebrates (2). These well-characterized molecules include NCAMs (3), L1 family CAMs (4), and nectins (5-8). Whereas cadherins and integrins require divalent cations such as Ca 2ϩ or Mg 2ϩ for their adhesive activities (9), IgCAMs are usually Ca 2ϩ -or Mg 2ϩ -independent (10). Moreover, most IgCAMs have preferences for homophilic and/or heterophilic interactions (10). In combination with these interactions, IgCAMs promote a variety of cell-cell associations through cis interaction within the plane of the membranes and/or trans interaction across the membranes (2, 11). For instance, the heterophilic cis interaction between L1 and NCAM appears to enhance the homophilic trans-binding activity of L1 (12, 13). Nectins form cis-homodimers that undergo homophilic and heterophilic trans interactions with each other to mediate cell-cell adhesion (6,11).In this study, the biochemical properties and subcellular localization of TSLC1 were investigated in the cells expressing TSLC1 tagged with GFP or endogenous TSLC1. We report the physiological properties of TSLC1 along with several lines of evidence that TSLC1 is a single transmembrane glycoprotein involved in cell-cell aggregation through homophilic trans interaction.

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We recently identified TSLC1, a tumor suppressor gene in human lung cancer. Gene silencing by promoter methylation has been observed frequently in adenocarcinoma of the lung, liver, and pancreas. Here, we demonstrate that TSLC1 expression is also absent or markedly reduced in 3 of 4 prostate cancer cell lines. Promoter sequences of TSLC1 were heavily methylated in PPC-1 cells that lacked TSLC1 expression, supporting the idea that promoter methylation is strongly correlated with complete loss of gene expression. Promoter sequences of TSLC1 were also methylated significantly in 7 of 22 (32%) primary prostate cancers. Hypermethylation of the promoter occurred not only in advanced tumors, but also in relatively early-stage tumors. Restoration of TSLC1 expression substantially suppressed tumor formation of PPC-1 cells in nude mice. These findings indicate that alteration of TSLC1 is involved in prostate cancer.

We have recently identi®ed the TSLC1 gene as a novel tumor suppressor in human non-small cell lung cancers. TSLC1 encodes a membrane glycoprotein with an extracellular domain homologous to those of immunoglobulin superfamily proteins. Truncation of TSLC1 in the cytoplasmic domain in a primary human tumor suggests that this domain is important for tumor suppressor activity. Here, we report the isolation of two TSLC1-like genes, TSLL1 and TSLL2, based on their structural homology with the sequences corresponding to the cytoplasmic domain of TSLC1. Signi®cant similarity was also observed in the extracellular domain as well as in the overall gene structure, indicating that these three genes form a unique subfamily (the TSLC1-gene family) in the immunoglobulin superfamily genes. In contrast to the ubiquitous expression of TSLC1, TSLL1 is expressed exclusively in adult and fetal human brain, while TSLL2 is expressed in several speci®c tissues including prostate, brain, kidney and some other organs. Expression of TSLL1 and TSLL2 was lost or markedly reduced in many human glioma cell lines or some prostate cancer cell lines, suggesting that loss of expression of these genes might be involved in some human cancers. Oncogene (2001) 20, 5401 ± 5407.

We have previously identified the tumor suppressor in lung cancer 1 (TSLC1) gene as a novel tumor suppressor in human non-small cell lung cancer (NSCLC) by functional complementation. TSLC1 encodes a membrane glycoprotein belonging to an immunoglobulin superfamily and participates in cell adhesion. A truncating mutation of the TSLC1 corresponding to its cytoplasmic domain in a primary NSCLC tumor suggests that this domain is important for tumor suppressor activity. Here, we report that TSLC1 directly associates with MPP3, one of the human homologues of a Drosophila tumor suppressor gene, Discs large (Dlg). This interaction was dependent on the presence of a PDZ-binding motif at the carboxyl terminus of TSLC1. Furthermore, TSLC1 and MPP3 were colocalized at the cell-cell attachment sites in both a low and a high cell density. The MPP3 gene was expressed in normal lung as well as in many tissues examined except for peripheral blood lymphocytes but lost its expression in one of the nine NSCLC cell lines. These results suggest that TSLC1 and MPP3 are involved in the same cascade of cell-cell interaction, and that the disruption of this cascade might lead cells to malignant growth and tumor formation in lung cancer.

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