Disruption of spermatogenic cell adhesion and male infertility in mice lacking TSLC1/IGSF4, an immunoglobulin superfamily cell adhesion molecule.

TSLC1/IGSF4, an immunoglobulin superfamily molecule, is predominantly expressed in the brain, lungs, and testes and plays important roles in epithelial cell adhesion, cancer invasion, and synapse formation. We generated Tslc1/Igsf4-deficient mice by disrupting exon 1 of the gene and found that Tslc1(-/-) mice were born with the expected Mendelian ratio but that Tslc1(-/-) male mice were infertile. In 11-week-old adult Tslc1(-/-) mice, the weight of a testis was 88% that in Tslc1(+/+) mice, and the number of sperm in the semen was approximately 0.01% that in Tslc1(+/+) mice. Histological analysis revealed that the round spermatids and the pachytene spermatocytes failed to attach to the Sertoli cells in the seminiferous tubules and sloughed off into the lumen with apoptosis in the Tslc1(-/-) mice. On the other hand, the spermatogonia and the interstitial cells, including Leydig cells, were essentially unaffected. In the Tslc1(+/+) mice, TSLC1/IGSF4 expression was observed in the spermatogenic cells from the intermediate spermatogonia to the early pachytene spermatocytes and from spermatids at step 7 or later. These findings suggest that TSLC1/IGSF4 expression is indispensable for the adhesion of spermatocytes and spermatids to Sertoli cells and for their normal differentiation into mature spermatozoa.

Promoter methylation of DAL-1/4.1B predicts poor prognosis in non-small cell lung cancer.

PURPOSE: DAL-1/4.1B is an actin-binding protein originally identified as a molecule whose expression is down-regulated in lung adenocarcinoma. We have previously shown that a lung tumor suppressor, TSLC1, associates with DAL-1, suggesting that both proteins act in the same cascade. The purpose of this study is to understand the molecular mechanisms and clinical significance of DAL-1 inactivation in lung cancer. EXPERIMENTAL DESIGN: We studied aberration of the DAL-1 in 103 primary non-small cell lung cancers (NSCLC) and 18 lung cancer cells. Expression and allelic and methylation status of DAL-1 was examined by reverse transcription-PCR, microsatellite analysis, and bisulfite sequencing or bisulfite single-strand conformational polymorphism, respectively. RESULTS: Loss of DAL-1 expression was strongly correlated with promoter methylation in lung cancer cells, whereas DAL-1 expression was restored by a demethylating agent, 5-aza-2'-deoxycytidine. The DAL-1 promoter was methylated in 59 (57%) primary NSCLC tumors, 37% of which were associated with loss of heterozygosity around the DAL-1 on chromosomal region 18p11.3. In squamous cell carcinomas, DAL-1 methylation was observed in 9 of 10 tumors at stage I, whereas the incidence of methylation gradually increased in adenocarcinomas as they progressed [13 of 36 (36%), 4 of 12 (33%), 14 of 17 (82%), and 3 of 3 (100%) tumors at stages I, II, III, and IV, respectively; P = 0.0026]. Furthermore, in adenocarcinomas, disease-free survival and overall survival were significantly shorter in patients with tumors harboring the methylated DAL-1 (P = 0.0011 and P = 0.045, respectively). CONCLUSIONS: DAL-1 methylation is involved in the development and progression of NSCLC and provides an indicator for poor prognosis.

Isolation of the mouse Tsll1 and Tsll2 genes, orthologues of the human TSLC1-like genes 1 and 2 (TSLL1 and TSLL2).

We have recently identified the human TSLL1 and TSLL2 genes, which are highly homologous to the human lung tumor suppressor gene, TSLC1. Loss of expression of the TSLL1 or TSLL2 in several cancers suggests that these genes could also act as tumor suppressors. Here, we report the isolation of the mouse orthologous genes, Tsll1 and Tsll2. The Tsll1 and Tsll2 cDNAs contain a single open reading frame of 1188 and 1164 bp encoding a putative immunoglobulin-like cell adhesion molecules of 396 and 388 amino acids, which display 95% and 98% identity with those of human TSLL1 and TSLL2, respectively. The Tsll1 and Tsll2 genes are both composed of nine exons and mapped on mouse chromosome 1q H2-H4 and on 7q A3-B2, respectively, both of which conserve syntenies with human chromosomes 1q and 19q. Like the human TSLL1, the mouse Tsll1 was expressed exclusively in the brain and neurogenic cells, while Tsll1 expression was lost in one of four rodent neuroblastoma cell lines. Tsll2 was expressed in the brain and several organs including the kidney and liver, whereas loss of Tsll2 expression was detected in some rodent cancer cells derived from these tissues. Furthermore, both murine TSLL1 and TSLL2 proteins were expressed on the plasma membrane, especially at the cell-cell attached site. These data, together with their strong conservation during the vertebrate evolution, suggest that TSLL1and TSLL2 could play an important role in cell-cell interaction as well as in tumor suppression.

Promoter hypermethylation of the potential tumor suppressor DAL-1/4.1B gene in renal clear cell carcinoma.

Renal clear cell carcinoma (RCCC) is a malignant tumor with poor prognosis caused by the high incidence of metastasis to distal organs. Although metastatic RCCC cells frequently show aberrant cytoskeletal organization, the underlying mechanism has not been elucidated. DAL-1/4.1B is an actin-binding protein implicated in the cytoskeleton-associated processes, while its inactivation is frequently observed in lung and breast cancers and meningiomas, suggesting that 4.1B is a potential tumor suppressor. We studied a possible involvement of 4.1B in RCCCs and evaluated it as a clinical indicator. 4.1B protein was detected in the proximal convoluted tubules of human kidney, the presumed cell of origin of RCCC. On the other hand, loss or marked reduction of its expression was observed in 10 of 19 (53%) renal cell carcinoma (RCC) cells and 12 of 19 (63%) surgically resected RCCC by reverse transcription-PCR. Bisulfite sequencing or bisulfite SSCP analyses revealed that the 4.1B promoter was methylated in 9 of 19 (47%) RCC cells and 25 of 55 (45%) surgically resected RCCC, and inversely correlated with 4.1B expression (p < 0.0001). Aberrant methylation appeared to be a relatively early event because more than 40% of the tumors with pT1a showed hypermethylation. Furthermore, 4.1B methylation correlated with a nuclear grade (p = 0.017) and a recurrence-free survival (p = 0.0036) and provided an independent prognostic factor (p = 0.038, relative risk 10.5). These results indicate that the promoter methylation of the 4.1B is one of the most frequent epigenetic alterations in RCCC and could predict the metastatic recurrence of the surgically resected RCCC.

Tumor suppressor in lung cancer (TSLC)1 suppresses epithelial cell scattering and tubulogenesis.

The tumor suppressor in lung cancer 1 (TSLC1/IGSF4) encodes an immunoglobulin-superfamily cell adhesion molecule whose cytoplasmic domain contains a protein 4.1-binding motif (protein 4.1-BM) and a PDZ-binding motif (PDZ-BM). Loss of TSLC1 expression is frequently observed in advanced cancers implying its involvement in tumor invasion and/or metastasis. Using Madin-Darby canine kidney cells expressing a full-length TSLC1 or various cytoplasmic deletion mutants of TSLC1, we examined the role of TSLC1 in epithelial mesenchymal transitions during the hepatocyte growth factor (HGF)-induced tubulogenesis and cell scattering. In a three-dimensional culture, the full-length TSLC1, which was localized to the lateral membrane of Madin-Darby canine kidney cysts, inhibited HGF-induced tubulogenesis. In contrast, the mutants lacking either the protein 4.1-BM or the PDZ-BM abolished the inhibitory effect on tubulogenesis. In addition, these mutants showed aberrant subcellular localization indicating that lateral localization is correlated with the effect of TSLC1. In a two-dimensional culture, the full-length TSLC1, but not the mutants lacking the protein 4.1-BM or the PDZ-BM, suppressed HGF-induced cell scattering. Furthermore, the cells expressing full-length TSLC1 retained E-cadherin-based cell-cell adhesion even after being treated with HGF. These cells showed prolonged activation of Rac and low activity of Rho, whereas the HGF-treated parental cells induced transient activation of Rac and sustained activation of Rho. Prolonged Rac activation caused by the expression of TSLC1 required its cytoplasmic tail. These findings, taken together, suggest that TSLC1 plays a role in suppressing induction of epithelial mesenchymal transitions by regulating the activation of small Rho GTPases.