DAL-1 Expression Research Articles

Expression, DNA methylation pattern and transcription factor EPB41L3 in gastric cancer: a study of 262 cases

PurposeDNA methylation prominently inactivates tumor suppressor genes and facilitates oncogenesis. Previously, we delineated a chromosome 18 deletion encompassing the erythrocyte membrane protein band 4.1-like 3 (EPB41L3) gene, a progenitor for the tumor suppressor that is differentially expressed in adenocarcinoma of the lung-1 (DAL-1) in gastric cancer (GC).MethodsOur current investigation aimed to elucidate EPB41L3 expression and methylation in GC, identify regulatory transcription factors, and identify affected downstream pathways. Immunohistochemistry demonstrated that DAL-1 expression is markedly reduced in GC tissues, with its downregulation serving as an independent prognostic marker.ResultsHigh-throughput bisulfite sequencing of 70 GC patient tissue pairs revealed that higher methylation of non-CpGs in the EPB41L3 promoter was correlated with more malignant tumor progression and higher-grade tissue classification. Such hypermethylation was shown to diminish DAL-1 expression, thus contributing to the malignancy of GC phenotypes. The DNA methyltransferase inhibitor 5-aza-2’-deoxycytidine (5-aza-CdR) was found to partially restore DAL-1 expression. Moreover, direct binding of the transcription factor CDC5L to the upstream region of the EPB41L3 promoter was identified via chromosome immunoprecipitation (ChIP)-qPCR and luciferase reporter assays. Immunohistochemistry confirmed the positive correlation between CDC5L and DAL-1 protein levels. Subsequent RNA-seq analysis revealed that DAL-1 significantly influences the extracellular matrix and space-related pathways. GC cell RNA-seq post-5-Aza-CdR treatment and single-cell RNA-seq data of GC tissues confirmed the upregulation of AREG and COL17A1, pivotal tumor suppressors, in response to EPB41L3 demethylation or overexpression in GC epithelial cells.ConclusionIn conclusion, this study elucidates the association between non-CpG methylation of EPB41L3 and GC progression and identifies the key transcription factors and downstream molecules involved. These findings enhance our understanding of the role of EPB41L3 in gastric cancer and provide a solid theoretical foundation for future research and potential clinical applications.

DAL-1/4.1B promotes the uptake of exosomes in lung cancer cells via Heparan Sulfate Proteoglycan 2 (HSPG2).

DAL-1/4.1B is frequently absent in lung cancer tissues, which is significantly related to the occurrence and development of lung cancer. In this research, we found that DAL-1/4.1B affected the uptake of exosomes by lung cancer cells. When the expression of DAL-1/4.1B increased and decreased, the ability of exosome uptake enhanced and attenuated correspondingly. And we found that when cells were treated with different vesicles uptake inhibitors (chlorpromazine, methyl-β-cyclodextrin (MβCD), cytochalasin D, chloroquine and heparin) and heparinase (HSPE), only heparin and HSPE counteracted the uptake enhancement effect caused by DAL-1/4.1B. Therefore, we speculated that DAL-1/4.1B might promote the uptake of exosomes through the heparan sulfate proteoglycans (HSPGs) pathway. After screening the expression of HSPGs and HSPE in H292 cells, the expression of heparan sulfate proteoglycan 2 (HSPG2) increased with overexpression of DAL-1/4.1B and decreased with knockdown of DAL-1/4.1B. Meanwhile, exosome uptake decreased with HSPG2 knockdown in H292 and DAL-1/4.1B-overexpressing H292 cells. Moreover, knockdown of DAL-1/4.1B and HSPG2 in lung cancer A549 cells resulted in a similar decrease in exosome uptake, and the expression of HSPG2 was also decreased with DAL-1/4.1B knockdown. These results indicated that HSPG2 directly affected the uptake of exosomes, while DAL-1/4.1B positively affected the expression of HSPG2. Therefore, DAL-1/4.1B may promote cellular adhesion and inhibit migration in cancer cells.

Decreased expression levels of DAL-1 and TOB1 are associated with clinicopathological features and poor prognosis in gastric cancer

PurposeWe previously demonstrated that the functional inactivation of DAL-1 and TOB1 promotes an aggressive phenotype in gastric cancer cells, but the links between both genes and the survival of patients with gastric cancer are unknown. Here, we investigated the correlations of the expression levels of DAL-1 and TOB1 with the progression of gastric cancer. MethodsA total of 270 patients who underwent resectable gastrectomy were included. The expression of DAL-1 and TOB1 was detected by immunohistochemistry. ResultsLow expression of DAL-1 in cancer tissue was significantly associated with tumor site (p < 0.05), histological grade (p < 0.01), depth of invasion (p < 0.05), lymph node metastasis status (p < 0.05), Lauren classification (p < 0.001), and clinical stage (p < 0.01). A lower level of TOB1 was observed in gastric cancer patients with diffuse type disease compared to patients with either intestinal or mixed type disease (p < 0.001). Additionally, Spearman’s correlation analysis revealed that decreased expression of DAL-1 was positively correlated with low TOB1 expression (r=0.304, p < 0.001). The survival analysis showed that low levels of DAL-1 and TOB1 were significantly associated with poor survival of gastric cancer patients (p <0.001 and p < 0.05, respectively). ConclusionThe downregulation of DAL-1 and TOB1 expression is associated with shorter survival of gastric cancer patients. Hence, DAL-1 and TOB1 may be considered potential novel markers for predicting the outcomes of patients with gastric cancer.

Aberrant expression of the candidate tumor suppressor gene DAL-1 due to hypermethylation in gastric cancer.

By allelotyping for loss of heterozygosity (LOH), we previously identified a deletion region that harbors the candidate tumor suppressor gene DAL-1 at 18p11.3 in sporadic gastric cancers (GCs). The expression and function of DAL-1 in GCs remained unclear. Here, we demonstrated that the absence of or notable decreases in the expression of DAL-1 mRNA and protein was highly correlated with CpG hypermethylation of the DAL-1 promoter in primary GC tissues and in GC cell lines. Furthermore, abnormal DAL-1 subcellular localization was also observed in GC cells. Exogenous DAL-1 effectively inhibited cancer cell proliferation, migration, invasion and epithelial to mesenchymal transition (EMT); exogenous DAL-1 also promoted apoptosis in GC AGS cells. When endogenous DAL-1 was knocked down in GC HGC-27 cells, the cells appeared highly aggressive. Taken together, these findings provide solid evidence that aberrant expression of DAL-1 by hypermethylation in the promoter region results in tumor suppressor gene behavior that plays important roles in the malignancy of GCs. Understanding the role of it played in the molecular pathogenesis of GC, DAL-1 might be a potential biomarker for molecular diagnosis and evaluation of the GC.

Loss of expression of the differentially expressed in adenocarcinoma of the lung (DAL-1) protein is associated with metastasis of non-small cell lung carcinoma cells

The differentially expressed in adenocarcinoma of the lung-1 (DAL-1) protein is a member of the membrane-associated cytoskeleton protein 4.1 family. This protein was previously found to be downregulated or lost in more than half of primary non-small cell lung cancers (NSCLC). In this study, the relationship between DAL-1 expression and NSCLC metastasis was examined. DAL-1 mRNA and protein levels were measured in NSCLC cell lines and in tumor cells isolated from the pleural fluid of NSCLC patients clinically diagnosed with distant metastases to the bone or brain. The results revealed that DAL-1 expression was observed in two (GLC-82 and NCI-H460) out of seven metastatic NSCLC cell lines examined. DAL-1 expression was not observed in the cells isolated from the pleural fluid in nine out of ten patients. Overexpression of DAL-1 in A549 cells, a cell line lacking endogenous DAL-1, inhibited cell migration and invasion by approximately 38 and 48 %, respectively. In contrast, DAL-1 knockdown in NCI-H460 cells enhanced the migration and invasion potential of this cell line 4.6- and 3-fold, respectively. Furthermore, DAL-1 promoter methylation was observed in six of nine pleural fluid NSCLC cell isolates and in two cell lines (A549 and H1299), as evidenced by a lack of endogenous DAL-1. Demethylation in A549 cells successfully restored DAL-1 mRNA and protein expression levels, resulting in a parallel remarkable inhibition of migration and invasion. These results indicated that DAL-1 was pivotal in triggering NSCLC migration and invasion and that loss of DAL-1 expression was due to the epigenetic methylation.

Relationship between Downregulation of DAL-1 and Carcinogenesis of Colorectal Adenocarcinoma

Objective To study the relationship of DAL-1 downregulation and carcinogenesis of colorectal adenocarcinoma.Methods RT-PCR was used to detect DAL-1 mRNA expression in colorectal cancer cell lines and colorectal adenocarcinoma tissues.BSP and MSP were performed to examine the DAL-1 promoter methylation status in colorectal cancer cell lines and colorectal adenocarcinoma tissues.5-aza-dC treatment was performed to verify the promoter methylation status of DAL-1 in colorectal cancer cell lines.Results Expression of DAL-1 was found in colorectal cancer cell lines;there was no significant difference between DAL-1 expressions among the cell lines.DAL-1 was significantly reduced in a portion of colorectal adenocarcinoma tissues.Frequency of DAL-1 downregulation was significantly higher in patients with lymph node metastasis or in Dukes C and Dukes D(P0.05).DAL-1 promoter methylation was rare phenomenon.Conclusion Downregulation of DAL-1 plays an important role in carcinogenesis of colorectal adenocarcinoma,especially in advanced colorectal adenocarcinoma.However,the gene promoter methylation doses not regulate the expression of DAL-1.

Arabidopsis DAL1 and DAL2, two RING finger proteins homologous to Drosophila DIAP1, are involved in regulation of programmed cell death

Programmed cell death (PCD) is a precise, genetically controlled cellular process with important roles in plant growth, development, and response to biotic and abiotic stress. However, the genetic mechanisms that control PCD in plants are unclear. Two Arabidopsis genes, DAL1 and DAL2 (for Drosophila DIAP1 like 1 and 2), encoding RING finger proteins with homology to DIAP1 were identified, and a series of experiments were performed to elucidate their roles in the regulation of PCD and disease resistance. Expression of DAL1 and DAL2 genes was induced in Arabidopsis plants after inoculation with virulent and avirulent strains of Pseudomonas syrinage pv. tomato (Pst) DC3000 or after infiltration with fumonisin B1 (FB1). Plants with mutations in the DAL1 and DAL2 genes displayed more severe disease after inoculation with an avirulent strain of Pst DC3000, but they showed similar disease severity as the wild-type plant after inoculation with a virulent strain of Pst DC3000. Significant accumulations of reactive oxygen species (ROS) and increased cell death were observed in the dal1 and dal2 mutant plants after inoculation with the avirulent strain of Pst DC3000. The dal mutant plants underwent extensive PCD upon infiltration of FB1 and displayed higher levels of ROS accumulation, callose deposition, and autofluorescence than the wild-type plants. Our data suggest that DAL1 and DAL2 may act as negative regulators of PCD in Arabidopsis.

Frequent concomitant epigenetic silencing of the stress‐responsive tumor suppressor gene CADM1, and its interacting partner DAL‐1 in nasal NK/T‐cell lymphoma

Nasal NK/T-cell lymphoma (NL) is a rare but clinically important entity of lymphoma. Its preferential incidence in Orientals but not Caucasians suggests possible genetic predisposition. 11q deletion is common in NL, indicating certain tumor suppressor genes (TSGs) at this locus involved in its pathogenesis. We investigated the expression and methylation of an 11q23.2 TSG, CADM1 (or TSLC1), and its partner DAL-1 (or EPB41L3) in NL. Methylation and silencing of CADM1 were detected in 2 NL and 4 of 8 (50%) of non-Hodgkin lymphoma (NHL) cell lines, but not in normal NK cells and normal PBMC. Absence of CADM1 protein was also detected in NL cell lines. 5-aza-2'-deoxycytidine (Aza) demethylation or genetic knockout of both DNMT1 and 3B genes restored CADM1 and DAL-1 expression. CADM1 methylation was further detected in 36 of 45 (80%) of NL tumors. Concomitantly, DAL-1 was epigenetically inactivated in NL cell lines and virtually all the tumors with methylated CADM1. A significant correlation between the methylation of both genes was found (p < 0.0001). Homozygous deletion of CADM1 was detected in only 3 of 18 (17%) of tumors. The stress-response of CADM1 was abolished when its promoter becomes methylated. Our results demonstrate a frequent, predominant epigenetic silencing of CADM1 and DAL-1 in NL, which likely play a synergic role in NL pathogenesis.

Hypermethylation of the Dal‐1 gene in lung adenocarcinomas induced by N‐nitrosobis(2‐hydroxypropyl)amine in rats

DAL-1 (differentially expressed in adenocarcinoma of the lung) is an actin-binding protein that has been shown to suppress growth in lung cancer cells. Recently, inactivation of the gene encoding DAL-1 due to hypermethylation has been found in several human malignancies, including lung cancers. To assess the involvement of the Dal-1 gene in rat lung carcinogenesis, we investigated the expression of Dal1 and its methylation status in rat lung adenocarcinomas induced by N-nitrosobis(2-hydroxypropyl)amine (BHP). Six-week old male Wistar rats (n = 11) were given 2,000 ppm BHP in their drinking water for 12 wk and maintained without further treatment until they were sacrificed at 25 wk. Total RNA was extracted from 11 lung adenocarcinomas, one from each BHP treated rat, and Dal-1 gene expression was analyzed using real-time quantitative reverse transcription-polymerase chain reaction. Dal-1 expression was significantly reduced in the lung adenocarcinomas compared with three normal lung tissues (P < 0.05). For methylation analysis, bisulfite sequencing was performed using normal lung tissue and tissue from 4 tumors, all of which showed reduced expression of Dal-1. The 5' upstream region was highly methylated in all four adenocarcinomas, whereas this region was unmethylated in normal lung tissue. These results suggest that aberrant methylation of the Dal-1 gene might be involved in the development of lung adenocarcinomas induced in rats by BHP.

Downregulation of TSLC1 and DAL-1 expression occurs frequently in breast cancer

TSLC1 and DAL-1 are tumor suppressor genes involved in cell adhesion. In this study, we examined the expression and methylation pattern of these genes in breast cancer cell lines and primary breast carcinomas. TSLC1 expression was lost in 5 of 8 (63%) and DAL-1 expression was lost in 6 of 8 (75%) breast cancer cell lines, respectively. Downregulation of TSLC1 expression was observed in 43 of 50 (86%) and of DAL-1 expression in 26 of 55 (47%) primary breast carcinomas. TSLC1 methylation was found in 4 of 8 (50%) and DAL-1 methylation was observed in 6 of 8 (75%) breast cancer cell lines, respectively. Of 95 primary breast carcinomas 46 (48%) were TSLC1 methylated and 26 (27%) were DAL-1 methylated. Twenty of 43 (47%) and 10 of 26 (38%) primary breast cancer samples which showed downregulation of TSLC1 and DAL-1 expression were unmethylated for these genes. Re-expression of TSLC1 and DAL-1 was observed after treatment of BT-20 cells with 5-aza-2'-deoxycytidine and TSA. Samples from patients with grade 3 tumors were more frequently TSLC1 and TSLC1 and/or DAL-1 methylated than samples from patients with grade 1 and 2 tumors (P = 0.032, P = 0.023). Moreover, TSLC1 methylation correlated with loss of both ER and PgR staining (P = 0.011, P = 0.02). Our findings suggest that TSLC1 and DAL-1 are involved in the pathogenesis of breast cancer and are frequently inactivated by methylation.

Loss of the Putative Tumor Suppressor Band 4.1B/Dal1 Gene Is Dispensable for Normal Development and Does Not Predispose to Cancer

The band 4.1 proteins are cytoskeletal proteins, harboring a conserved FERM domain highly homologous to the N-terminal FERM domain of ezrin, radixin, moesin, and merlin. Recently, a truncated form of the 4.1B protein, termed Dal-1, was identified in a screen as down regulated in adenocarcinoma of the lung and was mapped to chromosome 18p11.3, which is lost in 38% of primary non-small cell lung carcinoma tumors. Analysis of several meningiomas has shown that Dal-1 expression was lost in 76% of the tumors. To further elucidate the function of the 4.1B/Dal-1 gene in development and tumorigenesis we generated mice deficient for this allele. The 4.1B/Dal-1 null mice develop normally and are fertile. Rates of cellular proliferation and apoptosis in brain, mammary, and lung tissues from the 4.1B/Dal-1 null mice were indistinguishable from those seen with wild-type mice. Aging studies indicate that these mice do not have a propensity to develop tumors. Analysis of fibroblasts from these mice demonstrated that the growth characteristics and kinetics of these cells were not different from those of cells from the wild-type mice. These findings indicate that the 4.1B gene is not required for normal development and that 4.1B/Dal-1 does not function as a tumor suppressor gene.

Expression and methylation pattern of TSLC1 cascade genes in lung carcinomas

TSLC1 (tumor suppressor in lung cancer-1, IGSF4) encodes a member of the immunoglobulin superfamily molecules, which is involved in cell-cell adhesion. TSLC1 is connected to the actin cytoskeleton by DAL-1 (differentially expressed in adenocarcinoma of the lung-1, EPB41L3) and it directly associates with MPP3, one of the human homologues of a Drosophila tumor suppressor gene, Discs large. Recent data suggest that aberrant promoter methylation is important for TSLC1 inactivation in lung carcinomas. However, little is known about the other two genes in this cascade, DAL-1 and MPP3. Thus, we investigated the expression and methylation patterns of these genes in lung cancer cell lines, primary lung carcinomas and nonmalignant lung tissue samples. By reverse transcription-polymerase chain reaction, loss of TSLC1 expression was observed in seven of 16 (44%) non-small-cell lung cancer (NSCLC) cell lines and in one of 11 (9%) small-cell lung cancer (SCLC) cell lines, while loss of DAL-1 expression was seen in 14 of 16 (87%) NSCLC cell lines and in four of 11 (36%) SCLC cell lines. By contrast, MPP3 expression was found in all tumor cell lines analysed. Similar results were obtained by microarray analysis. TSLC1 methylation was seen in 13 of 39 (33%) NSCLC cell lines, in one of 11 (9%) SCLC cell lines and in 100 of 268 (37%) primary NSCLCs. DAL-1 methylation was observed in 17 of 39 (44%) NSCLC cell lines, in three of 11 (27%) SCLC cell lines and in 147 of 268 (55%) primary NSCLCs. In tumors of NSCLC patients with stage II-III disease, DAL-1 methylation was seen at a statistically significant higher frequency compared to tumors of patients with stage I disease. A significant correlation between loss of expression and methylation of the genes in lung cancer cell lines was found. Overall, 65% of primary NSCLCs had either TSLC1 or DAL-1 methylated. Methylation of one of these genes was detected in 59% of NSCLC cell lines; however, in SCLC cell lines, methylation was much less frequently observed. The majority of nonmalignant lung tissue samples was not TSLC1 or DAL-1 methylated. Re-expression of TSLC1 and DAL-1 was seen after treatment of lung cancer cell lines with 5-aza-2'-deoxycytidine. Our results suggest that methylation of TSLC1 and/or DAL-1, leading to loss of their expression, is an important event in the pathogenesis of NSCLC.

Promoter Methylation of DAL-1/4.1B Predicts Poor Prognosis in Non–Small Cell Lung Cancer

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. 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. 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). DAL-1 methylation is involved in the development and progression of NSCLC and provides an indicator for poor prognosis.

Immunolocalization of protein 4.1B/DAL-1 during neoplastic transformation of mouse and human intestinal epithelium

Recently, we have reported that the protein 4.1B immunolocalization occurred only in matured columnar epithelial cells of normal rat intestines. This finding suggested that protein 4.1B expression could be examined for a possible change during neoplastic transformation of the intestinal mucosa. In the present study, we first present the distribution of mouse protein 4.1B in normal intestinal epithelial cells and tumor cells using the adenomatous polyposis coli (Apc) mutant mouse model. A low level of protein 4.1B expression coincided with the phenotypic transition to carcinoma. To examine the protein 4.1B expression in human intestinal mucosa, we used another antibody against an isoform of the human protein 4.1B, DAL-1 (differentially expressed adenocarcinoma of the lung). Human DAL-1 was also expressed in matured epithelial cells in human colons, with a definite expression gradient along the crypt axis. In human colorectal cancer cells, however, DAL-1 expression was not detected. These results suggest that mouse protein 4.1B and human DAL-1 might have a striking analogy of functions, which may be integrally involved in epithelial proliferation. We propose that loss of protein 4.1B/DAL-1 expression might be a marker of intestinal tumors, indicative of a tumor suppressor function in the intestinal mucosa.

The MADS‐box gene DAL1 is a potential mediator of the juvenile‐to‐adult transition in Norway spruce (Picea abies)

Progression through the plant life cycle involves change in many essential features, most notably in the capacity to reproduce. The transition from a juvenile vegetative and non-reproductive to an adult reproductive phase is gradual and can take many years; in the conifer Norway spruce, Picea abies, typically 20-25 years. We present a detailed analysis of the activities of three regulatory genes with potential roles in this transition in Norway spruce: DAL1, a MADS-box gene related to the AGL6 group of genes from angiosperms, and the two LEAFY-related genes PaLFY and PaNLY. DAL1 activity is initiated in the shoots of juvenile trees at an age of 3-5 years, and then increases with age, whereas both LFY genes are active throughout the juvenile phase. The activity of DAL1 further shows a spatial pattern along the stem of the tree that parallels a similar gradient in physiological and morphological features associated with maturation to the adult phase. Constitutive expression of DAL1 in transgenic Arabidopsis plants caused a dramatic attenuation of both juvenile and adult growth phases; flowers forming immediately after the embryonic phase of development in severely affected plants. Taken together, our results support the notion that DAL1 may have a regulatory role in the juvenile-to-adult transition in Norway spruce.

DAL-1/4.1B tumor suppressor interacts with protein arginine N-methyltransferase 3 (PRMT3) and inhibits its ability to methylate substrates in vitro and in vivo.

DAL-1 (differentially expressed in adenocarcinoma of the lung)/4.1B is a tumor suppressor gene on human chromosome 18p11.3 whose expression is lost in >50% of primary non-small-cell lung carcinomas. Based on sequence similarity, DAL-1/4.1B has been assigned to the Protein 4.1 superfamily whose members interact with plasma membrane proteins through their N-terminal FERM (4.1/Ezrin/Radixin/Moesin) domain, and cytoskeletal components via their C-terminal SAB (spectrin-actin binding) region. Using the DAL-1/4.1B FERM domain as bait for yeast two-hybrid interaction cloning, we identified protein arginine N-methyltransferase 3 (PRMT3) as a specific DAL-1/4.1B-interacting protein. PRMT3 catalyses the post-translational transfer of methyl groups from S-adenosyl-L-methionine to arginine residues of proteins. Coimmunoprecipitation experiments using lung and breast cancer cell lines confirmed this interaction in mammalian cells in vivo. In vitro binding assays demonstrated that this was an interaction occurring via the C-terminal catalytic core domain of PRMT3. DAL-1/4.1B was determined not to be a substrate for PRMT3-mediated methylation but its presence inhibits the in vitro methylation of a glycine-rich and arginine-rich methyl-accepting protein, GST (glutathione-S-transferase-GAR (glycine- and arginine-rich), which contains 14 'RGG' consensus methylation sites. In addition, induced expression of DAL-1/4.1B in MCF-7 breast cancer cells showed that the DAL-1/4.1B protein significantly inhibits PRMT3 methylation of cellular substrates. These findings suggest that modulation of post-translational methylation may be an important mechanism through which DAL-1/4.1B affects tumor cell growth.

Disruption of 14-3-3 binding does not impair Protein 4.1B growth suppression.

Meningiomas are common central nervous system tumors; however, the mechanisms underlying their pathogenesis are largely unknown. Collaborative studies from our laboratory demonstrated a direct association of 14-3-3 with the meningioma tumor suppressor Protein 4.1B, which was not observed with other members of the Protein 4.1 family, including the NF2 meningioma tumor suppressor, merlin/schwannomin. Given the role of 14-3-3 in the regulation of cell proliferation and apoptosis, we sought to determine the functional significance of 14-3-3 binding to Protein 4.1B growth suppression. Based on comparative binding studies performed with additional members of the Protein 4.1 family, we generated specific missense mutations within the minimal growth suppressor fragment of Protein 4.1B (DAL-1, differentially expressed in adenocarcinoma of the lung). Complementary in vitro GST affinity chromatography and in vivo interaction experiments demonstrated that the F359Y mutation abrogated binding to 14-3-3, but did not impair DAL-1 binding to other known Protein 4.1B interacting proteins. Similar to wild-type DAL-1, the expression of the F359Y DAL-1 14-3-3-binding mutant resulted in reduced Protein 4.1B-deficient IOMM-Lee and CH157-MN meningioma cell line colony formation. Moreover, similar to wild-type DAL-1, the stable expression of the DAL-1 F359Y mutant significantly reduced cell proliferation in independently isolated IOMM-Lee clones, as assessed by thymidine incorporation. Collectively, these results suggest that binding to 14-3-3 is not essential for the growth suppressor function of Protein 4.1B in meningiomas.

Suppression of growth and increased cellular attachment after expression of DAL-1 in MCF-7 breast cancer cells.

The differentially expressed in adenocarcinoma of the lung (DAL-1) gene, which shares significant homology with members of the 4.1/ezrin/radixin/moesin/neurofibromatosis 2 (ERM/NF2) protein family, has previously been shown to suppress growth in lung cancer cell lines. This gene localizes to chromosome band 18p11.3, which undergoes loss of heterozygosity (LOH) in nonsmall cell lung carcinomas and a significant proportion of ductal carcinomas in situ (DCIS) of the breast. This finding suggests that alteration of gene(s) (possibly DAL-1) within this chromosomal region may be important early in the progression of breast disease. We generated MCF-7 cell lines expressing DAL-1 constitutively or under the control of an inducible promoter and analyzed the effect of DAL-1 expression on growth. These investigations revealed that the DAL-1 protein suppresses the growth of MCF-7 cells and may do so in part through the induction of apoptosis. In addition, expression of DAL-1 increased attachment of these cells to a variety of extracellular matrices. This is the first evidence that the DAL-1 protein functions at the interface between cell adhesion and apoptosis in controlling cell growth.

Merlin, DAL-1, and progesterone receptor expression in clinicopathologic subsets of meningioma: a correlative immunohistochemical study of 175 cases.

The molecular pathogenesis of meningiomas is poorly characterized. Loss of NF2 (merlin) expression has been reported in 30%-80% of all sporadic meningiomas. Recently, we found that loss of expression for a second Protein 4.1-family tumor suppressor. DAL-1, is also common. A biologically important role for progesterone receptor (PR) has also been proposed based on its reported inverse relationship with tumor grade. In order to better define the pathogenetic roles of these proteins, we studied the merlin, DAL-1, and PR immunoprofiles in 175 fully characterized meningiomas, including nonrecurring versus recurring benign, proliferative versus brain invasive atypical and anaplastic subtypes. Loss of expression for either Protein 4.1-family tumor suppressor (merlin or DAL-1) was almost universal (92%), with combined losses being common (58%). Individually, absence of merlin or DAL-1 protein was detected in 74% and 76% respectively, with no significant differences among the 5 subsets. PR immunoreactivity was commonly associated with retained DAL-1 expression (p < 0.001) and with tumor grade, with 51% of benign, 21% of atypical, and 11% of anaplastic tumors staining positive (p < 0.001). We conclude that PR immunohistochemistry may have diagnostic utility in meningothelial neoplasms. Protein 4.1-family tumor suppressor losses are likely important early events in meningioma pathogenesis, whereas PR expression is associated with benignity.

Loss of DAL-1, a protein 4.1-related tumor suppressor, is an important early event in the pathogenesis of meningiomas.

Meningiomas are common nervous system tumors, whose molecular pathogenesis is poorly understood. To date, the most frequent genetic alteration detected in these tumors is loss of heterozygosity (LOH) on chromosome 22q. This finding led to the identification of the neurofibromatosis 2 (NF2) tumor suppressor gene on 22q12, which is inactivated in 40% of sporadic meningiomas. The NF2 gene product, merlin (or schwannomin), is a member of the protein 4.1 family of membrane-associated proteins, which also includes ezrin, radixin and moesin. Recently, we identified another protein 4.1 gene, DAL-1 (differentially expressed in adenocarcinoma of the lung) located on chromosome 18p11.3, which is lost in approximately 60% of non-small cell lung carcinomas, and exhibits growth-suppressing properties in lung cancer cell lines. Given the homology between DAL-1 and NF2 and the identification of significant LOH in the region of DAL-1 in lung, breast and brain tumors, we investigated the possibility that loss of expression of DAL-1 was important for meningioma development. In this report, we demonstrate DAL-1 loss in 60% of sporadic meningiomas using LOH, RT-PCR, western blot and immunohistochemistry analyses. Analogous to merlin, we show that DAL-1 loss is an early event in meningioma tumorigenesis, suggesting that these two protein 4.1 family members are critical growth regulators in the pathogenesis of meningiomas. Furthermore, our work supports the emerging notion that membrane-associated alterations are important in the early stages of neoplastic transformation and the study of such alterations may elucidate the mechanism of tumorigenesis shared by other tumor types.