The Health History of First-Degree Relatives' Dyslipidemia Can Affect Preferences and Intentions following the Return of Genomic Results for Monogenic Familial Hypercholesterolemia.

Genetic testing is key in modern healthcare, particularly for monogenic disorders such as familial hypercholesterolemia. This Tohoku Medical Megabank Project study explored the impact of first-degree relatives' dyslipidemia history on individual responses to familial hypercholesterolemia genomic results. Involving 214 participants and using Japan's 3.5KJPN genome reference panel, the study assessed preferences and intentions regarding familial hypercholesterolemia genetic testing results. The data revealed a significant inclination among participants with a family history of dyslipidemia to share their genetic test results, with more than 80% of participants intending to share positive results with their partners and children and 98.1% acknowledging the usefulness of positive results for personal health management. The study underscores the importance of family health history in genetic-testing perceptions, highlighting the need for family-centered approaches in genetic counseling and healthcare. Notable study limitations include the regional scope and reliance on questionnaire data. The study results emphasize the association between family health history and genetic-testing attitudes and decisions.

Trans-homophilic interaction of CADM1 promotes organ infiltration of T-cell lymphoma by adhesion to vascular endothelium.

The initial step of organ infiltration of malignant cells is the interaction with host vascular endothelial cells, which is often mediated by specific combinations of cell adhesion molecules. Cell adhesion molecule 1 (CADM1) is overexpressed in adult T-cell leukemia/lymphoma (ATL) and provides a cell-surface diagnostic marker. CADM1 promotes the adhesion of ATL cells to vascular endothelial cells and multiple organ infiltration in mice. However, its binding partner on host cells has not yet been identified. In this study, we show that CADM1 promotes transendothelial migration of ATL cells in addition to the adhesion to vascular endothelial cells. Moreover, CADM1 enhances liver infiltration of mouse T-cell lymphoma cells, EL4, after tail vein injection, whereas a CADM1 mutant lacking adhesive activity did not. Among the known CADM1-binding proteins expressed in primary endothelial cells, only CADM1 and CADM4 could induce morphological extension of ATL cells when plated onto glass coated with these proteins. Furthermore, CADM1-mediated liver infiltration of EL4 cells was canceled in conventional and vascular endothelium-specific Cadm1 knockout mice, whereas it was not canceled in Cadm4 knockout mice. These results suggest that CADM1 on host vascular endothelial cells is required for organ infiltration of ATL and other T-cell lymphomas expressing CADM1.

The return of individual genomic results to research participants: design and pilot study of Tohoku Medical Megabank Project.

Certain large genome cohort studies attempt to return the individual genomic results to the participants; however, the implementation process and psychosocial impacts remain largely unknown. The Tohoku Medical Megabank Project has conducted large genome cohort studies of general residents. To implement the disclosure of individual genomic results, we extracted the potential challenges and obstacles. Major challenges include the determination of genes/disorders based on the current medical system in Japan, the storage of results, prevention of misunderstanding, and collaboration of medical professionals. To overcome these challenges, we plan to conduct multilayer pilot studies, which deal with different disorders/genes. We finally chose familial hypercholesterolemia (FH) as a target disease for the first pilot study. Of the 665 eligible candidates, 33.5% were interested in the pilot study and provided consent after an educational "genetics workshop" on the basic genetics and medical facts of FH. The genetics professionals disclosed the results to the participants. All positive participants were referred to medical care, and a serial questionnaire revealed no significant psychosocial distress after the disclosure. Return of genomic results to research participants was implemented using a well-prepared protocol. To further elucidate the impact of different disorders, we will perform multilayer pilot studies with different disorders, including actionable pharmacogenomics and hereditary tumor syndromes.

Identification of two novel breast cancer loci through large-scale genome-wide association study in the Japanese population.

Genome-wide association studies (GWAS) have successfully identified about 70 genomic loci associated with breast cancer. Owing to the complexity of linkage disequilibrium and environmental exposures in different populations, it is essential to perform regional GWAS for better risk prediction. This study aimed to investigate the genetic architecture and to assess common genetic risk model of breast cancer with 6,669 breast cancer patients and 21,930 female controls in the Japanese population. This GWAS identified 11 genomic loci that surpass genome-wide significance threshold of P < 5.0 × 10-8 with nine previously reported loci and two novel loci that include rs9862599 on 3q13.11 (ALCAM) and rs75286142 on 21q22.12 (CLIC6-RUNX1). Validation study was carried out with 981 breast cancer cases and 1,394 controls from the Aichi Cancer Center. Pathway analyses of GWAS signals identified association of dopamine receptor medicated signaling and protein amino acid deacetylation with breast cancer. Weighted genetic risk score showed that individuals who were categorized in the highest risk group are approximately 3.7 times more likely to develop breast cancer compared to individuals in the lowest risk group. This well-powered GWAS is a representative study to identify SNPs that are associated with breast cancer in the Japanese population.

Induction of the Matrix Metalloproteinase 13 Gene in Bronchial Epithelial Cells by Interferon and Identification of its Novel Functional Polymorphism.

Matrix metalloproteinases (MMPs) are a class of extra-cellular and membrane-bound proteases involved in a wide array of physiological and pathological processes including tissue remodeling, inflammation, and cytokine secretion and activation. MMP-13 has been shown to be involved in lung diseases such as acute lung injury, viral infections, and chronic obstructive pulmonary disease; however, the molecular pathogenesis of MMP-13 in these conditions is not well understood. In this study, we investigated the mechanisms and roles of MMP-13 secretion in human small airway epithelial cells (SAECs) and functional polymorphisms of the MMP13 gene. Polyinosinic-polycytidylic acid (poly(I:C)) and interferon ß (IFN-ß) stimulated the secretion of MMP-13 from SAECs by more than several hundred-fold. Stimulation of the secretion by poly(I:C) was abolished by SB304680 (p38 inhibitor), LY294002 (PI3K inhibitor), Janus kinase (JAK) inhibitor I, RNA-activated protein kinase (PKR) inhibitor, and Bay 11-7082 (NF-κB inhibitor), while stimulation by IFN-ß was inhibited by all except Bay 11-7082. These data suggested that the secretion of MMP-13 was mediated through IFN receptor pathways independently of nuclear factor kappa B (NF-κB) and that poly(I:C) stimulated IFN secretion in an NF-κB-dependent manner from SAECs, leading to IFN-stimulated MMP-13 secretion. Chemical MMP-13 inhibitors and MMP-13 small interfering RNA (siRNA) inhibited IFN-stimulated secretion of interferon gamma-inducible protein 10 (IP-10) and regulated on activation, normal T-cell expressed and secreted (RANTES), suggesting that MMP-13 is involved in the secretion of these virus-induced proinflammatory chemokines. We identified a novel functional polymorphism in the promoter region of the MMP13 gene. The MMP13 gene may play important roles in defense mechanisms of airway epithelial cells.

Dynamic regulation of a cell adhesion protein complex including CADM1 by combinatorial analysis of FRAP with exponential curve-fitting.

Protein components of cell adhesion machinery show continuous renewal even in the static state of epithelial cells and participate in the formation and maintenance of normal epithelial architecture and tumor suppression. CADM1 is a tumor suppressor belonging to the immunoglobulin superfamily of cell adhesion molecule and forms a cell adhesion complex with an actin-binding protein, 4.1B, and a scaffold protein, MPP3, in the cytoplasm. Here, we investigate dynamic regulation of the CADM1-4.1B-MPP3 complex in mature cell adhesion by fluorescence recovery after photobleaching (FRAP) analysis. Traditional FRAP analysis were performed for relatively short period of around 10 min. Here, thanks to recent advances in the sensitive laser detector systems, we examine FRAP of CADM1 complex for longer period of 60 min and analyze the recovery with exponential curve-fitting to distinguish the fractions with different diffusion constants. This approach reveals that the fluorescence recovery of CADM1 is fitted to a single exponential function with a time constant (τ) of approximately 16 min, whereas 4.1B and MPP3 are fitted to a double exponential function with two τs of approximately 40-60 sec and 16 min. The longer τ is similar to that of CADM1, suggesting that 4.1B and MPP3 have two distinct fractions, one forming a complex with CADM1 and the other present as a free pool. Fluorescence loss in photobleaching analysis supports the presence of a free pool of these proteins near the plasma membrane. Furthermore, double exponential fitting makes it possible to estimate the ratio of 4.1B and MPP3 present as a free pool and as a complex with CADM1 as approximately 3:2 and 3:1, respectively. Our analyses reveal a central role of CADM1 in stabilizing the complex with 4.1B and MPP3 and provide insight in the dynamics of adhesion complex formation.

Trans-homophilic interaction of CADM1 activates PI3K by forming a complex with MAGuK-family proteins MPP3 and Dlg.

CADM1 (Cell adhesion molecule 1), a cell adhesion molecule belonging to the immunoglobulin superfamily, is involved in cell-cell interaction and the formation and maintenance of epithelial structure. Expression of CADM1 is frequently downregulated in various tumors derived from epithelial cells. However, the intracellular signaling pathways activated by CADM1-mediated cell adhesion remain unknown. Here, we established a cell-based spreading assay to analyze the signaling pathway specifically activated by the trans-homophilic interaction of CADM1. In the assay, MDCK cells expressing exogenous CADM1 were incubated on the glass coated with a recombinant extracellular fragment of CADM1, and the degree of cell spreading was quantified by measuring their surface area. Assay screening of 104 chemical inhibitors with known functions revealed that LY294002, an inhibitor of phosphoinositide 3-kinase (PI3K), efficiently suppressed cell spreading in a dose-dependent manner. Inhibitors of Akt and Rac1, downstream effectors of PI3K, also partially suppressed cell spreading, while the addition of both inhibitors blocked cell spreading to the same extent as did LY294002. Furthermore, MPP3 and Dlg, membrane-associated guanylate kinase homologs (MAGuK) proteins, connect CADM1 with p85 of PI3K by forming a multi-protein complex at the periphery of cells. These results suggest that trans-homophilic interaction mediated by CADM1 activates the PI3K pathway to reorganize the actin cytoskeleton and form epithelial cell structure.

Genomic and transcriptional alterations of cholangiocarcinoma.

Cholangiocarcinoma (CCA) is one of the representative cancers refractory to any therapeutic approach. The incidence of CCA is highest in the northeastern part of Thailand, where chronic inflammation caused by liver fluke (Opisthorchis viverrini: Ov) infection is a major etiologic factor. The incidence of CCA is also increasing in other countries, including Japan. Here, we overview the genetic and transcriptional alterations of CCA with and without association with Ov infection. CCA with Ov shows enhanced expression of the genes involved in xenobiotic metabolism and chronic inflammatory responses, including cytokine signaling, whereas CCA without Ov shows enhanced expression of growth factor signaling, such as HER2. Exome and the following prevalence sequencing identified mutations of the BAP1, ARID1A, IDH1 and IDH2 genes in CCA, in addition to the high incidence of known mutations in the TP53, KRAS2 SMAD4, and CDKN2A genes, suggesting the role of chromatin modulators in CCA pathogenesis. CCA with Ov shows significantly higher incidence of the TP53 gene mutation, whereas CCA without Ov showed significantly more frequent mutations of the BAP1, IDH1 and IDH2 genes. However, CCAs with Ov and without Ov share a similar mutation spectrum dominated by C : G > T : A transitions mainly at CpG dinucleotides, suggesting that CCA shares etiologic factors with pancreatic ductal carcinoma but not with hepatocellular carcinoma. Comprehensive analyses of the genetic and transcriptional alterations of CCA with and without Ov infection would provide useful information for the prevention, early diagnosis, and treatment of CCA.

Trans-homophilic interaction of CADM1 activates PI3K by forming a complex with MAGuK-family proteins MPP3 and Dlg.

CADM1 (Cell adhesion molecule 1), a cell adhesion molecule belonging to the immunoglobulin superfamily, is involved in cell-cell interaction and the formation and maintenance of epithelial structure. Expression of CADM1 is frequently down-regulated in various tumors derived from epithelial cells. However, the intracellular signaling pathways activated by CADM1-mediated cell adhesion remain unknown. Here, we established a cell-based spreading assay to analyze the signaling pathway specifically activated by the trans-homophilic interaction of CADM1. In the assay, MDCK cells expressing exogenous CADM1 were incubated on the glass coated with a recombinant extracellular fragment of CADM1, and the degree of cell spreading was quantified by measuring their surface area. Assay screening of 104 chemical inhibitors with known functions revealed that LY294002, an inhibitor of phosphoinositide 3-kinase (PI3K), efficiently suppressed cell spreading in a dose-dependent manner. Inhibitors of Akt and Rac1, downstream effectors of PI3K, also partially suppressed cell spreading, while the addition of both inhibitors blocked cell spreading to the same extent as did LY294002. Furthermore, MPP3 and Dlg, membrane-associated guanylate kinase homologs (MAGuK) proteins, connect CADM1 with p85 of PI3K by forming a multi-protein complex at the periphery of cells. These results suggest that trans-homophilic interaction mediated by CADM1 activates the PI3K pathway to reorganize the actin cytoskeleton and form epithelial cell structure.

Expression of a splicing variant of the CADM1 specific to small cell lung cancer.

CADM1, a member of the immunoglobulin superfamily cell adhesion molecule, acts as a tumor suppressor in a variety of human cancers. CADM1 is also ectopically expressed in adult T-cell leukemia (ATL), conferring an invasive phenotype characteristic to ATL. Therefore, CADM1 plays dual roles in human oncogenesis. Here, we investigate the roles of CADM1 in small cell lung cancer (SCLC). Immunohistochemistry demonstrates that 10 of 35 (29%) primary SCLC tumors express CADM1 protein. Western blotting and RT-PCR analyses reveal that CADM1 is significantly expressed in 11 of 14 SCLC cells growing in suspension cultures but in neither of 2 SCLC cells showing attached growth to plastic dishes, suggesting that CADM1 is involved in anchorage-independent growth in SCLC. In the present study, we demonstrate that SCLC expresses a unique splicing variant of CADM1 (variant 8/9) containing additional extracellular fragments corresponding to exon 9 in addition to variant 8, a common isoform in epithelia. Variant 8/9 of CADM1 is almost exclusively observed in SCLC and testis, although this variant protein localizes along the membrane and shows similar cell aggregation activity to variant 8. Interestingly, both variant 8/9 and variant 8 of CADM1 show enhanced tumorigenicity in nude mice when transfected into SBC5, a SCLC cell lacking CADM1. Inversely, suppression of CADM1 expression by shRNA reduced spheroid-like cell aggregation of NCI-H69, an SCLC cell expressing a high amount of CADM1. These findings suggest that CADM1 enhances the malignant features of SCLC, as is observed in ATL, and could provide a molecular marker specific to SCLC.

Aberrant expression of tumor suppressors CADM1 and 4.1B in invasive lesions of primary breast cancer.

BACKGROUND: The tumor suppressor genes CADM1/TSLC1 and DAL-1/4.1B are frequently inactivated by promoter methylation in non-small cell lung cancer. The proteins they encode, CADM1 and 4.1B, form a complex in human epithelial cells and are involved in cell-cell adhesion. METHODS: Expression of CADM1 and 4.1B proteins was examined by immunohistochemistry in 67 primary breast cancer and adjacent noncancerous tissues. CADM1 and 4.1B messenger RNA (mRNA) was detected by reverse-transcription polymerase chain reaction (RT-PCR). The methylation status of the CADM1 and 4.1B promoters was determined quantitatively by bisulfite treatment followed by pyrosequencing. RESULTS: CADM1 and 4.1B protein signals were detected along the cell membrane in normal mammary epithelia. By contrast, 47 (70%) and 49 (73%) of 67 primary breast cancers showed aberrant CADM1 and 4.1B staining, respectively. Aberrant CADM1 staining was more frequently observed in pT2 and pT3 tumors and for stages II and III (P = 0.045 and P = 0.020, respectively), while aberrant 4.1B staining was more often observed in tumors with lymph node metastasis, for pT2 and pT3 tumors, and for stages II and III (P = 0.0058, P = 0.0098, and P = 0.0007, respectively). Furthermore, aberrant CADM1 and 4.1B expression was preferentially observed in invasive relative to noninvasive lesions from the same specimen (P = 0.036 and P = 0.0009, respectively). Finally, hypermethylation of CADM1 and 4.1B genes was detected in 46% and 42% of primary breast cancers, respectively. CONCLUSIONS: Our findings suggest that aberrant CADM1 and 4.1B expression is involved in progression of breast cancer, especially in invasion into the stroma and metastasis.

Aberrations of a cell adhesion molecule CADM4 in renal clear cell carcinoma.

Renal clear cell carcinoma (RCCC) is the most frequent subpopulation of renal cell carcinoma and is derived from the proximal uriniferous tubules. We have previously reported that an actin-binding protein, 4.1B/DAL-1, is expressed in renal proximal tubules, whereas it is inactivated in 45% of RCCC by promoter methylation. In the lung and several epithelial tissues, 4.1B is shown to associate with a tumor suppressor protein, CADM1, belonging to the immunoglobulin-superfamily cell adhesion molecules. Here, we demonstrate by immunohistochemistry that another member of the CADM-family protein, CADM4, as well as 4.1B is expressed specifically in human proximal tubules, while CADM1 and 4.1N, another member of the 4.1 proteins, are expressed in the distal tubules. Immunoprecipitation analysis coupled with Western blotting revealed that CADM4 associated with 4.1B, while CADM1 associated with 4.1N in the lysate from normal human kidney, implicating that a cascade of CADM4 and 4.1B plays an important role in normal cell adhesion of the proximal tubules. On the other hand, CADM4 expression was lost or markedly reduced in 7 of 10 (70%) RCC cell lines and 28 of 40 (70%) surgically resected RCCC, including 10 of 16 (63%) tumors with T1a. CADM4 expression was more preferentially lost in RCCC with vascular infiltration (p = 0.04), suggesting that loss of CADM4 is involved in tumor invasion. Finally, introduction of CADM4 into an RCC cell line, 786-O, dramatically suppressed tumor formation in nude mice. These findings suggest that CADM4 is a novel tumor suppressor candidate in RCCC acting with its binding partner 4.1B.

Transcriptional regulation of the CADM1 gene by retinoic acid during the neural differentiation of murine embryonal carcinoma P19 cells.

CADM1 is a multifunctional cell adhesion molecule expressed predominantly in the nerve system, testis and lung. The expression of the Cadm1 gene is induced during the neural differentiation of murine embryonal carcinoma P19 cells by treatment with retinoic acid (RA). Here, we show that the suppression of CADM1 expression using RNAi interfered with P19 cell aggregation and reduced cell populations expressing MAP2 after RA treatment. Nonaggregated P19 cells were not differentiated into neurons, suggesting that CADM1 participates in the aggregate formation and neuronal differentiation of P19 in vitro. A luciferase assay of a series of deletion mutants of the CADM1 promoter localized an RA-responsive cis-acting element to an approximately 90-bp fragment upstream of the translational start site. This element contains a putative binding site for transcription factor Sp1, named Sp1-binding site-1 (Sp1BS-1). Sp1BS-1 and adjacent Sp1-binding sites (Sp1BS-2 and Sp1BS-3) showed enhanced transcriptional activity by RA. Moreover, a chromatin immunoprecipitation showed that RA receptor (RAR)α was associated with a DNA fragment containing Sp1BS-1, whereas suppression of RARα expression using siRNA reduced the responsiveness of the CADM1 promoter to RA. These results suggest that Sp1 plays a critical role in RA-induced CADM1 expression through possible interaction with RARα in the neural differentiation of P19.

Tumor suppressor CADM1 is involved in epithelial cell structure.

The tumor suppressor, CADM1, is involved in cell adhesion and preferentially inactivated in invasive cancer. We have previously reported that CADM1 associates with an actin-binding protein, 4.1B/DAL-1, and a scaffold protein, membrane protein palmitoylated 3 (MPP3)/DLG3. However, underlying mechanism of tumor suppression by CADM1 is not clarified yet. Here, we demonstrate that MPP1/p55 and MPP2/DLG2, as well as MPP3, interact with both CADM1 and 4.1B, forming a tripartite complex. We then examined cell biological roles of CADM1 and its complex in epithelia using HEK293 cells. Among MPP1-3, MPP2 is recruited to the CADM1-4.1B complex in the early process of adhesion in HEK293 cells. By suppression of CADM1 expression using siRNA, HEK293 lose epithelia-like structure and show flat morphology with immature cell adhesion. 4.1B and MPP2, as well as E-cadherin and ZO-1, are mislocalized from the membrane by depletion of CADM1 in HEK293. Mislocalization of MPP2 is also observed in several cancer cells lacking CADM1 expression with the transformed morphology. These findings suggest that CADM1 is involved in the formation of epithelia-like cell structure with 4.1B and MPP2, while loss of its function could cause morphological transformation of cancer cells.

The interaction of IQGAP1 with the exocyst complex is required for tumor cell invasion downstream of Cdc42 and RhoA.

Invadopodia are actin-based membrane protrusions formed at contact sites between invasive tumor cells and the extracellular matrix with matrix proteolytic activity. Actin regulatory proteins participate in invadopodia formation, whereas matrix degradation requires metalloproteinases (MMPs) targeted to invadopodia. In this study, we show that the vesicle-tethering exocyst complex is required for matrix proteolysis and invasion of breast carcinoma cells. We demonstrate that the exocyst subunits Sec3 and Sec8 interact with the polarity protein IQGAP1 and that this interaction is triggered by active Cdc42 and RhoA, which are essential for matrix degradation. Interaction between IQGAP1 and the exocyst is necessary for invadopodia activity because enhancement of matrix degradation induced by the expression of IQGAP1 is lost upon deletion of the exocyst-binding site. We further show that the exocyst and IQGAP1 are required for the accumulation of cell surface membrane type 1 MMP at invadopodia. Based on these results, we propose that invadopodia function in tumor cells relies on the coordination of cytoskeletal assembly and exocytosis downstream of Rho guanosine triphosphatases.

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.

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.

Detection of allelic imbalance in the gene expression of hMSH2 or RB1 in lymphocytes from pedigrees of hereditary, nonpolyposis, colorectal cancer and retinoblastoma by an RNA difference plot.

A number of phenotypes in hereditary disorders or common diseases are associated with specific genotypes. However, little is known about the molecular basis of phenotypic variation among individuals carrying the same mutation or polymorphism. Here, a highly quantitative approach was taken to examine a relative amount of mRNA from two polymorphic alleles with a coefficient of variation of less than 10% using an RNA difference plot (RDP). RDP analysis revealed that most genes examined were expressed in equal amount from the two alleles in normal lymphocytes. In contrast, the relative amounts of hMSH2 or RB1 mRNAs carrying premature termination codons were significantly reduced compared with those of wild-type mRNAs in lymphocytes from carriers of hereditary, nonpolyposis, colorectal cancer and hereditary retinoblastoma. The balance of allelic expression of the RB1 was also significantly impaired in a pedigree of retinoblastoma carrying a missense mutation in codon 661. The relative expression of the mutant to the wild-type RB1 alleles among the carriers varied from 0.40 to 2.39. The analysis of the expression diversity of a disease-associated allele by RDP could provide a novel approach to elucidating the mechanisms underlying phenotypic variation among individuals carrying an identical mutation or polymorphism at a single locus.