Dab2 mRNA Research Articles

The effect of eight weeks of moderate and high intensity aerobic training on the gene expression of Mir-145, Wnt3a and Dab2 in the heart tissue of type 2 diabetic rats.

Pathological hypertrophy of heart tissue has been attributed to changes in some microRNAs and their target genes in heart tissue. This study intended to study the effects of eight weeks of moderate and high intensity aerobic training (MIT&HIT) on the mRNA of Mir-145, Wnt3a, and Dab2 in heart tissue of type 2 diabetic rats. To implement this experimental research, 60 male Wistar rats were randomly divided into 6 groups, including Healthy-control (HC), Diabetic-control (DC), Moderate intensity training (MIT), Diabetes-MIT (DMIT), high intensity training (HIT) and Diabetes-HIT (DHIT). The aerobic training was conducted with moderate (50-60% VO2max) and high (85-90% VO2max) intensity, 5days a week, for 8weeks. The Mir-145, Wnt3a and Dab2 gene expression in the heart tissue samples was measured by Real Time PCR. Data were analyzed by one-way ANOVA and Tukey post hoc test at the P < 0.05. Moderate and high intensity aerobic training was associated with non-significant increase in Mir-145 mRNA of Heart tissue in type 2 diabetic rats than the diabetic control group(P < 0.05). Moderate and high intensity aerobic training was associated with significant increase in Wnt3a mRNA (P = 0.001) and significant decrease in Dab-2 mRNA (P = 0.001) of Heart tissue in type 2 diabetic rats than the diabetic control group. The Dab-2 mRNA was significantly lower of heart tissue in the diabetes- high intensity training group than the diabetes- moderate intensity training group (P = 0.001). It seems that moderate and high intensity aerobic exercise can help regulate the genes of the physiological hypertrophy pathway of the heart tissue in diabetes.

Loss of Disabled-2 Expression in Pancreatic Cancer Progression

Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive cancer type characterized by rapid metastasis and resistance to chemotherapy, properties that are shared by cancer stem cells (CSCs). In pancreatic cancer, tumor cells which possess the properties of CSCs also phenotypically resemble cells that have undergone epithelial-to-mesenchymal transition or EMT. Disabled-2 (Dab2) is a multifunctional scaffold protein frequently downregulated in cancer that has been linked to the process of EMT. However, the role of Dab2 in pancreatic cancer development and progression remains unclear. Downregulation of Dab2 expression in pancreatic cancer cell lines was found to trigger induction of genes characteristic of EMT and the CSC phenotype, while overexpression of Dab2 in the Panc1 cell line blocked the process of TGFβ-stimulated EMT. In addition, selective inhibition of the TGFβRI/RII receptors was found to reverse genes altered by Dab2 downregulation. Dab2 mRNA expression was found to be decreased in PDAC tumor samples, as compared to levels observed in normal pancreatic tissue. Methylation of the Dab2 gene promoter was demonstrated in Stage I PDAC tumors and in the MiaPaCa2 cell line, suggesting that promoter methylation may silence Dab2 expression early in pancreatic cancer progression. These results suggest that Dab2 may function as a tumor suppressor in pancreatic cancer by modulation of the TGFβ-stimulated EMT and CSC phenotype.

Rapid Downregulation of DAB2 by Toll-Like Receptor Activation Contributes to a Pro-Inflammatory Switch in Activated Dendritic Cells.

Dendritic cells (DCs) are pivotal in regulating tolerogenic as well as immunogenic responses against microorganisms by directing both the innate and adaptive immune response. In health, phenotypically different DC subsets found in the gut mucosa are maintained in their tolerogenic state but switch to a pro-inflammatory phenotype during infection or chronic autoinflammatory conditions such as inflammatory bowel disease (IBD). The mechanisms that promote the switch among the mucosal DCs from a tolerogenic to an immunogenic, pro-inflammatory phenotype are incompletely understood. We hypothesized that disabled homolog 2 (DAB2), recently described as a negative regulator of DC immunogenicity during their development, is regulated during intestinal inflammation and modulates mucosal DC function. We show that DAB2 is highly expressed in colonic CD11b+CD103− DCs, a subset known for its capacity to induce inflammatory Th1/Th17 responses in the colon, and is downregulated predominantly in this DC subset during adoptive T cell transfer colitis. Administration of Dab2-deficient DCs (DC2.4Dab2−/− cells) modulated the course of DSS colitis in wild-type mice, enhanced mucosal expression of Tnfa, Il6, and Il17a, and promoted neutrophil recruitment. In bone-marrow derived dendritic cells (BMDC), DAB2 expression correlated with CD11b levels and DAB2 was rapidly and profoundly inhibited by TLR ligands in a TRIF- and MyD88-dependent manner. The negative modulation of DAB2 was biphasic, initiated with a quick drop in DAB2 protein, followed by a sustained reduction in Dab2 mRNA. DAB2 downregulation promoted a more functional and activated DC phenotype, reduced phagocytosis, and increased CD40 expression after TLR activation. Furthermore, Dab2 knockout in DCs inhibited autophagy and promoted apoptotic cell death. Collectively, our results highlight the immunoregulatory role for DAB2 in the intestinal dendritic cells and suggest that DAB2 downregulation after microbial exposure promotes their switch to an inflammatory phenotype.

MicroRNA-145 regulates disabled-2 and Wnt3a expression in cardiomyocytes under hyperglycaemia.

MicroRNA-145 (miR-145) could protect cardiomyocyte apoptosis against oxidative stress and repair infarcted myocardium. Angiotensin II (Ang II), a pro-inflammatory cytokine could modulate myocardial remodelling. However, the role of hyperglycaemia on miR-145 expression in cardiomyocyte or diabetes is not known. The effect of Ang II on miR-145 expression under hyperglycaemia in cardiomyocytes remains unknown. We sought to investigate the effect of hyperglycaemia and Ang II on miR-145 expression in cardiomyocytes. Rat cardiomyocytes were cultured under high glucose concentration (25mmol/L), and streptozotocin-induced diabetic rats were established. TaqMan® MicroRNA real-time quantitative assay was used to quantitate miR-145. Sustained high glucose concentration (hyperglycaemia) significantly decreased miR-145 expression in cardiomyocytes. Hyperglycaemia significantly increased Ang II mRNA expression and secretion from rat cardiomyocytes. Ang II suppressed miR-145 expression in cardiomyocytes. Hyperglycaemia increased Dab2 and decreased Wnt3a/ß-catenin expression in cardiomyocytes. Repression of miR-145 expression by Ang II resulted in increased Dab2 and decreased Wnt3a and ß-catenin expression under hyperglycaemia. In contrast, overexpression of miR-145 significantly decreased Dab2 mRNA and protein expression, whereas the mRNA and protein levels for Wnt3a and ß-catenin were significantly reduced in left ventricular myocardium from 5 days to 28days in diabetic rats. The protein expression patterns of Dab2 and Wnt3a/ß-catenin in left ventricular myocardium of diabetic rats could be reversed upon treatment with valsartan. Ang II downregulates miR-145 to regulate Dab2 and Wnt3a/ß-catenin expression in cardiomyocytes under high glucose concentration. Ang II plays a critical role in the regulation of miR-145 in cardiomyocytes under hyperglycaemic conditions.

Abstract 2382: Exploring the relationship between reelin signaling and breast cancer metastasis

Abstract We have investigated the role of reelin signaling in the progression of metastatic breast cancer. Reelin signaling serves to position neurons in the developing central nervous system, but functions of this signaling pathway in other tissues have not been studied until recently. Our laboratory discovered that the reelin pathway is an important regulator of mammary gland development. We showed that reelin and Dab1, the intracellular adaptor protein for reelin, are required for timely development and proper morphology of the mammary gland. We also found that normal mammary epithelial cells (MECs) can respond to exogenous reelin by slowing their migration rate. Since aberration of developmental pathways is often implicated in breast cancer progression, we studied the possible roles of the reelin pathway in this process. We found that reelin signaling pathway components are differentially expressed across a variety of human breast cancer cell lines. We also showed that mouse mammary carcinoma 4T1 cells do not respond to reelin by slowing their migration, likely through downregulation of Dab1 mRNA. Orthotopic injections of 4T1 cells into Balb/C mice are used to model stage IV triple negative breast cancer, and resulted in aggressive tumors that produced numerous metastases in the lungs by 4 weeks post-injection. In order to find whether reelin signaling functions in the tumor microenvironment, we injected 4T1 cells into mice that lack reelin or Dab1 (reeler and Dab1 mutant mice). We found that 4T1 tumor growth was delayed in reeler mutants, and we are currently in the process of assessing the metastatic burden in these mice. We did not see differences in tumor growth or metastatic lung colonization in Dab1 mutant mice. These results indicate that the reelin signaling pathway may promote tumor growth via Dab1-independent, non-canonical signaling events. Citation Format: Elvira Khialeeva, Joan Chou, Ellen M. Carpenter. Exploring the relationship between reelin signaling and breast cancer metastasis. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2382. doi:10.1158/1538-7445.AM2015-2382

Functional enrichment analysis of three Alzheimer's disease genome-wide association studies identities DAB1 as a novel candidate liability/protective gene

To explore genetic contributions of Alzheimer's disease (AD) at the level of biological terms and pathways, we analyzed three Caucasian population-based genome-wide association study datasets (TGEN_ND, GeneADA and NIA_LOAD) using the Database for Annotation, Visualization and Integrated Discovery (DAVID). This analysis identified 4 annotation terms (“Fibronectin type III-like fold,” “Cell adhesion,” “Cell motion” and “Ig-like-C2-type 3”) and 17 genes that associated with AD susceptibility in two or more of the GWAS datasets. Ten of these genes, have previously been identified as candidate AD liability genes in genetic association studies (AGT, COL11A1) or encode proteins that function in biological systems or pathways previously implicated in AD (BARHL2, CSF3R, DAB1, HMCN1, LEPR, PTPRF, PXDN, TNR). Among these, DAB1 (Dab, reelin signal transducer, homolog 1) was of particular interest, since it encodes a protein that functions downstream from reelin, a signaling pathway previously identified as protective in AD. Multiple linear regression analysis of correlations between brain DAB1 mRNA expression and SNP genotype using data from the “BrainCloud” database identified five SNPs within the DAB1 locus that correlated with mRNA expression in human dorsolateral prefrontal cortex. Analysis of predicted levels of DAB1 mRNA expression based on genotype combinations present in AD cases and controls vs. the log10-transformed odds ratios for AD diagnosis, revealed statistically significant correlations in one of the GWAS datasets (GenADA), with high DAB1 mRNA expression correlating with AD protection. Multidimensional scaling (MDS) analysis of cases and controls in the three GWAS, revealed genetic differences between GenADA and TGEN_ND/NIA_LOAD, which were similar to each other. To our knowledge, this study is the first to provide genetic evidence for DAB1 as a candidate AD liability/protection gene, although the strength of the contribution of DAB1 may differ among populations.

Expression of Dab2 in different adrenocortical adenomas

Objective To determine the expression and distribution of Disabled-2(Dab2) in normal human adrenal glands, and further to study the expression of Dab2 in tissues of different adrenocortical adenomas, and to elucidate whether Dab2 can be a specific molecular marker in the pathology of primary aldosteronism. Methods Real-time PCR and immunohistochemical staining were used to detect Dab2 expression in 10 aldosterone-producing adenoma (APA) samples, 8 cortisol-producing adenoma (CPA) samples, 8 non-functioning adenoma(NFA) samples and 6 normal adrenal samples. Results Immunohistochemical staining showed that Dab2 was significantly highly expressed in zona glomerulosa of normal human adrenal glands. Sporadical cluster of ZG cells with moderate Dab2 staining were demonstrated in APAs. In all CPA and NFA tumors, weak dab2 staining was detected. According to the results of real-time PCR, Dab2 mRNA expression was increased significantly in APAs compared with normal adrenal glands. There was no significant difference between normal adrenal glands, CPAs, and NFAs in regard to Dab2 mRNA expression. Compared to nontumor portions, APAs also showed higher Dab2 mRNA expression in the tumor(P<0.05). Conclusion Dab2 was predominantly localized in zona glomerulosa in normal adrenal gland. Increased Dab2 mRNA expression was detected in APAs compared with normal adrenal glands. Whereas, Dab2 protein expression was just moderate increased in APAs. Weather Dab2 can be a specific molecular marker in the pathology of primary aldosteronism has to be further studied. (Chin J Endocrinol Metab, 2015, 31: 127-132) Key words: Dab2; Primary aldosteronism; Molecular markers

Abstract B26: Reelin signaling in tumor microenvironment is required for metastasis of mammary carcinoma cells.

Abstract We have investigated the role of reelin signaling in the microenvironment of breast cancer and found that reelin is essential for progression of mammary tumors to metastasis. Reelin signaling serves to position neurons in the developing central nervous system, but functions of this signaling pathway in other tissues have not been studied until recently. Our laboratory discovered that the reelin pathway is an important regulator of mammary gland development. We showed that reelin and Dab1, the intracellular adaptor protein for reelin, are required for timely development and proper morphology of the mammary gland. We also found that normal mammary epithelial cells (MECs) can respond to exogenous reelin by slowing their migration rate, while mouse mammary carcinoma 4T1 cells lose this response, likely through downregulation of Dab1 mRNA. Orthotopic injections of 4T1 cells into wild type Balb/C mice resulted in aggressive tumors that produced numerous metastases in the lungs by 4 weeks post-injection. However, when these cells were injected into mice that lack reelin or Dab1 (reeler and Dab1 mutant mice), we saw a dramatic reduction in lung metastases despite the presence of primary tumors comparable in size to the tumors formed in wild type mice. We show that this phenotype is dependent upon loss of reelin in the host environment, since tumor cells isolated from reeler mutant mice produce primary tumors and lung metastases when injected into Balb/C mice. Using soft agar colony assays and Western blotting, we show that the inability of 4T1 cells to metastasize in mutant animals might be due to a reduced capacity of mammary carcinoma cells to undergo epithelial to mesenchymal transition (EMT) in an environment deficient for reelin signaling. In addition, tumors from reeler and Dab1 mutant mice appear to have more differentiated cells as shown by anti-α smooth muscle Actin and anti-K14 immunostaining. Ki-67 staining showed no differences in proliferation between tumors from wild type, reeler or Dab1 mutant mice. Finally, tail vein injections of 4T1 cells into reeler mutant mice also fail to produce metastases, indicating that reelin signaling in the host environment might have important functions at several steps of the metastatic process. Our results implicate reelin signaling as an essential component of transition from the primary tumor to a metastatic carcinoma. Citation Format: Elvira Khialeeva, Joan W. Chou, Ellen M. Carpenter. Reelin signaling in tumor microenvironment is required for metastasis of mammary carcinoma cells.. [abstract]. In: Abstracts: AACR Special Conference on Cellular Heterogeneity in the Tumor Microenvironment; 2014 Feb 26-Mar 1; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(1 Suppl):Abstract nr B26. doi:10.1158/1538-7445.CHTME14-B26

Vitamin D Transport Proteins Megalin and Disabled-2 Are Expressed in Prostate and Colon Epithelial Cells and Are Induced and Activated by All-Trans-Retinoic Acid

Megalin and disabled-2 (Dab2) are essential for uptake of the 25-hydroxycholecalciferol (25D3)-vitamin D binding protein (DBP) complex in tissues. In the kidney, this mechanism regulates serum 25D3 levels and production of 1,25-dihydroxycholecalciferol (1,25D3) by CYP27B1 for systemic use. Previously, we showed that mammary epithelial cells expressing CYP27B1 express megalin and Dab2 and internalize DBP by endocytosis, indicating 25D3 was accessible for conversion to 1,25D3 in extra-renal tissues. Moreover, induction of megalin and Dab2 (protein and mRNA abundance) by all-trans-retinoic acid (RA) enhanced DBP uptake. This suggests megalin and Dab2 play a central role in uptake of vitamin D and may predict actions of vitamin D in extra-renal tissues. Here, we characterized megalin and Dab2 expression and uptake of DBP in transformed human prostate and colon epithelial cells. Megalin and Dab2 were expressed in prostate and colon epithelial cells, which was markedly enhanced following treatment with RA. Furthermore, DBP uptake was stimulated by low-dose RA supplementation in LNCaP, PC-3, and Caco-2 cells. Taken together, these are the first studies to our knowledge that have demonstrated modulated expression of megalin and Dab2, as well as an association between increased expression of endocytic proteins with DBP uptake in prostate and colon cells.

Aberrant promoter methylation of Dab2 gene in myelodysplastic syndrome

Human Disabled-2 (Dab2), a putative tumor suppressor gene, is frequently down-regulated in human tumors. This study aims to explore the association between Dab2 methylation status and expression in newly diagnosed myelodysplastic syndrome (MDS) patients and patients who received 5-aza-2'-deoxycytidine (decitabine) treatment, so as to determine the effect of Dab2 in the pathogenesis of MDS. Methylation-specific polymerase chain reaction and bisulfite sequencing were used to detect the methylation status of Dab2 gene. Dab2 expression was investigated by using fluorescence quantitation RT-PCR (FQRT-PCR) and western blot analysis. Hypermethylation of Dab2 gene was present in 50.6% of patients with MDS and was significantly correlated with the down-regulation of Dab2 mRNA and protein expression. There was a significant difference in methylation frequency between refractory anemia/refractory anemia with ringed sideroblasts/MDS associated with isolated del (5q) (RA/RARS/5q-) group (33.3%) and refractory anemia with excess blasts-1/-2 (RAEB-1/RAEB-2) group (73.3%). Significant difference was also observed between refractory cytopenia with multiline dysplasia group (37.8%) and RAEB-1/RAEB-2 group. In addition, higher frequency of hypermethylation was observed in intermediate-2-/high-risk group, compared to low-risk/intermediate-1-risk group (75.0% vs. 40.0%). Demethylating agent 5-aza-2'-deoxycytidine treatment could partly reverse hypermethylation and, hence, restore the expression of Dab2 gene. The Dab2 gene is inactivated in MDS in part by DNA methylation, and the suppression of Dab2 expression by DNA methylation may play a role in the development of MDS.

Mechanisms by Which Estrogen Accelerates Ovarian Tumor Initiation.

Ovarian cancers present as three different types, epithelial, germ cell, and stromal, with epithelial cancers consisting ~90% of cases. As a result, it is believed that the majority of cancers that develop in the ovary arise from the ovarian surface epithelium. Previous studies have shown that exposure of mice to exogenous estradiol (E2) causes preneoplastic changes in ovarian surface epithelial (OSE) cell morphology and accelerates ovarian tumor onset. Clinically, estrogenic compounds are used for treatments such as hormone replacement therapy for relief of menopausal symptoms or as a form of contraceptive such as birth control pills. However, women using estrogen-only hormone replacement are at higher risk of ovarian cancer. Thus, the aim of this project is to determine the mechanisms by which estrogen has detrimental effects that promote an increased risk of ovarian cancer, specifically by elucidating the actions of E2 on the OSE. The pathways controlling the premalignant transformation are unknown, but exogenous E2 promote stratification and apparent loss of cellular polarity in the OSE layer. We hypothesized that this change in phenotype may be due to decreases in disabled-2 (Dab2), as Dab2 is required for epithelial cells to maintain polarity and a genome-wide screen has identified estrogen response elements in regions proximal to the transcriptional start site of Dab2 in mice and humans. Dab2 is an adaptor protein that is found in a variety of tissues and is highly expressed in breast and ovarian tissue but its expression is lost in the majority of ovarian carcinomas. To determine if Dab2 mediates the actions of E2, we examined the effects of E2 exposure on Dab2 expression (by qPCR) in cultures of normal mouse OSE (mOSE), SV40 T antigen-immortalized mOSE, and in a mouse ovarian cancer cell line (MASE). None of the cell lines showed a change in Dab2 expression in response to E2 over a range of doses (0-1000 nM) and treatment periods (0-72 hours). To determine the effects of E2 exposure on Dab2 expression in vivo, SCID mice were intraperitoneally injected with MASE cells and inserted subcutaneously with a 6o-day timed release 0.25 mg E2 pellet. Tumors showed a significant 8-fold decrease in Dab2 mRNA expression in response to E2 treatment. Tumors from the tgCAG-LS-TAg transgenic model of ovarian cancer also showed a trend for E2 suppression of Dab2 expression. Immunohistochemical detection of Dab2 shows that it is present in normal human and mouse OSE, but is lost in areas of stratified mOSE that occur at higher incidence after 60 days of exogenous E2 treatment. These results demonstrate that E2 exposure decreases Dab2 expression in both OSE and ovarian cancers in vivo, but not in vitro. The lack of response in vitro suggests that growing OSE cells on plastic is not a good model to study changes in mOSE polarity, a result that is consistent with previous studies using mammary epithelial cells. Current experiments are investigating alternative culture systems to develop a better model for the study of OSE cell polarity and epithelial cell architecture.

Reelin signalling in neuroblastoma: Migratory switch in metastatic stages

The essential functions of Reelin for the migratory behaviour of neuroblasts in the central nervous system are well documented. Its role in the dissemination of neuronal tumours of the peripheral nervous system has not been studied in detail. Here, we examined neuroblastoma (NB), a tumour derived from sympathoadrenal cells of neural crest origin. We studied the expression of Reelin, its receptors VLDLR and LRP8 and the adapter protein DAB1 in primary tumour samples and cell lines. We used real-time RT-PCR, immunohistology and western blot analysis. In NB cell lines we studied effects of all-trans retinoic acid and the in vitro effects of Reelin. In primary tumour samples of untreated patients, a significant downregulation of Reelin and DAB1 mRNA was found in the metastatic stages 3, 4 and 4s. Immunohistochemical studies revealed expression of Reelin, LRP8 and DAB1 in differentiating-type low-grade NB. In vitro, western blot analysis of selected NB cell lines showed variable expression patterns. Differentiation induction with all-trans retinoic acid induced the upregulation of Reelin and DAB1. Reelin acted as a chemoattractant for various NB cell lines but inhibited migration when applied together with the NB cells. In normal tissue, we found Reelin in lymphatic endothelial cells (LECs) but not in blood vessel endothelium (BECs). In primary NB, both BECs and LECs were positive. Our data strongly suggest that Reelin has a dual role in NB. Autocrine expression marks low-grade differentiating tumour cells, whereas paracrine Reelin presented by LECs and BECs may act as a chemoattractant and promote hematogenic and lymphogenic dissemination in progressed stages.

Dab1 (Disable Homolog-1) Reelin Adaptor Protein Is Overexpressed in the Olfactory Bulb at Early Postnatal Stages

Dab1 mediates reelin signalling and plays critical roles in early brain development such as the stereotypical positioning of neurons in the brain. The olfactory bulb undergoes a prominent layering reorganization, but shows not apparent differences between wild type and reeler in the layer organization. Therefore, an accurate regional and cellular simultaneous analysis of these molecules becomes essential to clarify the role played by Dab1 upon Reelin effect. The present study reveals a strong and consistent Dab1 mRNA and protein expressions, throughout the olfactory bulb layers in both wild type and reeler mice. In addition, noteworthy is the pattern of Dab1 location within cell nuclei in both strains. Furthermore, a temporal increment of Dab1 expression levels is detected from P0 to P15 in both strains, being the protein quantity higher in reeler than in wild type mice. Altogether, our results revealed that Reln acts directly from projection neurons via the production of different Reln fragments. Changes in the pattern of Dab1 expression could reflect an alternative Reln function in postnatal and adult stages, besides a possible regulation of Dab1 by other molecules distinct to Reln.

Regulation of Dab2 expression in intestinal and renal epithelia by development

Disabled-2 (Dab2) is an intracellular adaptor protein proposed to function in endocytosis. Here, we investigate the intestinal and renal Dab2 expression versus maturation. Dab2 mRNA levels measured by RT-PCR are greater in the small than in the large intestine. Immunological studies localize Dab2 to the terminal web domain of the enterocytes and reveal the presence of a 96-kDa Dab2 isoform in the apical membrane of the jejunum, ileum, and renal cortex of the suckling and adult rat. A 69-kDa Dab2 isoform is only observed in the apical membranes of the suckling ileum. During the suckling period, the Dab2 mRNA levels measured in the enterocytes and crypts and those of the 96-kDa Dab2 isoform are greater in the ileum than in the jejunum. No segmental differences are observed in the adult intestine. In the intestine, the levels of Dab2 mRNA and those of the 96-kDa Dab2 isoform decrease to adult values at weaning, whereas in the kidney they increase with development. Weaning the pups on a commercial milk diet slows the periweaning decline in the levels of Dab2 mRNA in the crypts and of those of the 96-kDa isoform. This is the first report showing that the 96-kDa Dab2 isoform is expressed at the apical domain of rat small intestine, that ontogeny regulates Dab2 gene expression in intestine and kidney and that retarding weaning affects intestinal Dab2 gene expression.

Abstract 251: Disabled gene is involved in CML progression and its expression level at diagnosis can predict major molecular response (MMR) to imatinib therapy

Abstract Despite the role of Bcr-Abl in the pathogenesis of Chronic Myeloid Leukemia (CML) is well established, the mechanisms leading to CML progression remain unknown. Using our model of Drosophila melanogaster (Dm) transgenic for human Bcr-Abl we identified Dab1 and Dab2, the homologs of Dm Disabled (Dab), as genes involved in CML progression. In Dm the Dab loss of function induced a worsening of the hBcr-Abl eye phenotype and an even stronger phenotype was obtained using Dab RNAi fly strains. By contrast, Dab gain of function rescued Bcr-Abl phenotype. Dab is an adaptor protein acting downstream of many receptor tyrosine kinases (RTK). One of the human homolog of Dab, Dab1 is a large common fragile site gene involved in neural migration, and the other homolog Dab2 encodes an adaptor protein implicated in RTK signalling, endocytosis, cell adhesion and differentiation. The downregulation of both genes is described in many cancers suggesting their possible role in oncogenesis but their involvement in haematological malignancies has never been described. The aim of the study was to investigate the role of Dab1/2 in CML progression. Dab1 and Dab2 mRNA was analyzed by Real Time PCR in 94 samples from 82 CML patients (34 PB and 60 BM) distributed as follows: 55 patients at diagnosis (19 enrolled in TOPS study), 9 chronic phase (CP), 7 accelerated phase (AP) and 11 blast crisis (BC). 21 healthy donors (10 PB and 11 BM) were analyzed as control. In 18 patients, genes expression was analyzed during remission as well. Protein expression was evaluated by Western Blot (WB) and Immunofluorescence (IF). In addition, K562 cells were transfected with Dab plasmids to evaluate the effects on cell proliferation. We found that in CML patients Dab1/2 expression was significantly decreased both in BM and PB (p&amp;lt;0.002 and p&amp;lt;0.0004) compared to healthy donors. In BC Dab1/2 levels were further decreased whereas during remission the expression was comparable to normal values. Data analysis of patients included in TOPS studies shows that Dab1 values are higher among those achieving MMR by 12 months (median value: 0,017) compared to those without MMR (median value: 0,001); WB and IF confirmed the absence of Dab1/2 proteins in course of active CML samples while it reappeared during remission. Moreover, Dab1 transfection of K562 significantly reduced proliferation (p=0,002). In conclusion, our results show a significant decrease of Dab1/2 expression in BC samples, when compared to CP CML and healthy donors. Among CP CML patients the responders to Tyrosine Kinase Inhibitors (TKI) therapy have been detected to express higher Dab levels than non responders, and these expression levels can predict MMR to Imatinib therapy. In conclusion, this study points to specific gene pathways that might offer new molecular markers for the monitoring of CML and new targets for CML therapy in order to prevent or overcome disease progression. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 251.

Megalin‐ and Disabled‐2‐mediated uptake of vitamin D‐binding protein is modulated by all‐trans‐retinoic acid in prostate and colon epithelial cells

Disabled‐2 (Dab2) is an adapter protein, that along with the membrane receptors megalin and cubilin, is essential for uptake of protein‐complexed 25‐hydroxycholecalciferol (25D3) in the kidney and maintenance of serum vitamin D homeostasis. Previously, we showed that mammary cells (T‐47D) also express Dab2, megalin, and cubilin and internalize vitamin D‐binding protein (DBP) by receptor‐mediated endocytosis. Moreover, induction of Dab2 and megalin by the differentiating agent all‐trans‐retinoic acid (RA) correlated with DBP uptake in mammary cells. These findings suggest that expression of Dab2, megalin, and cubilin are critical for the uptake and anti‐tumorigenic effects of vitamin D in cancer‐prone tissues. In this present study, we have extended our work by characterizing the uptake of DBP and the expression of Dab2 and megalin in prostate cancer (PC‐3 and LNCaP) and colon cancer (Caco‐2) cells. Our results indicate that Dab2 and megalin mRNA and protein are abundant in PC‐3, LNCaP, and Caco‐2 cell lines. This expression was markedly enhanced when treated with 10 μM RA. Furthermore, DBP was readily internalized by PC‐3, LNCaP, and Caco‐2 cells. Taken together, these are the first studies to our knowledge that have characterized a potential role for Dab2 and megalin in the uptake of vitamin D in prostate and colon cells. Supported by NIH SR03CA128091‐03 and Iowa State University Experiment station to MJR.

Rat small intestine expresses the reelin–Disabled‐1 signalling pathway

Expression of reelin, reelin receptors [apolipoprotein E receptor 2 (ApoER2) and very low density lipoprotein receptor (VldlR)] and the Disabled-1 (Dab1) protein was investigated in rat intestinal mucosa. Intestinal reelin and Dab1 mRNA levels were maximal in the early stages of life, reaching adult levels in 1-month-old rats. Expression of reelin mRNA was restricted to fibroblasts, whereas mRNAs of Dab1, ApoER2, VldlR and integrins alpha3 and beta1 were observed in enterocytes, crypts and fibroblasts. Reelin protein was only observed in isolated intestinal fibroblasts and in a cell layer subjacent to the villus epithelium, which seems to be composed of myofibroblasts because it also reacted to alpha-smooth muscle actin. The Disabled-1 and VldlR protein signals were detected in the crypt and villus cells, and they were particularly abundant in the terminal web domain of the enterocytes. The ApoER2 protein signal was detected in the upper half of the villi but not in the crypts. This is the first report showing that rat intestinal mucosa expresses the reelin-Disabled-1 signalling system.

Disabled Gene Is Involved in CML Progression and Its Expression Level at Diagnosis Can Predict Major Molecular Response (MMR) to Imatinib Therapy.

Abstract 3964Poster Board III-900Despite the role of Bcr-Abl in the pathogenesis of Chronic Myeloid Leukemia (CML) is well established, the mechanisms leading to CML progression remain unknown. By using our model of Drosophila melanogaster (Dm) transgenic for human Bcr-Abl we have identified Dab1 and Dab2 as genes involved in CML progression. In Dm the loss of function of these genes induced a worsening of the phenotype generated by hBcr-Abl expression. Moreover, an even stronger phenotype was obtained by silencing Disabled using RNAi fly strains. By contrast, Dab gain of function rescued Bcr-Abl phenotype. Disabled is a non receptor tyrosine kinase, identified in Dm as an adaptor protein acting downstream of many tyrosine kinases receptors (RTK). One of the human homolog of Disabled, Dab1 is a large common fragile site gene, involved in neural migration and the other homolog Dab2 codes is an adaptor protein implicated in RTK signalling, endocytosis, cell adhesion and differentiation. The downregulation of both genes is described in many types of cancer suggesting their possible role in oncogenesis but their involvement in haematological malignancies has never been described. The aim of the study was to investigate the role of Dab1/2 in CML progression. Dab1 and Dab2 mRNA was analyzed by Real Time PCR in 94 samples from 82 CML patients (34 PB and 60 BM). Among those patients 55 were collected at diagnosis (19 enrolled in TOPS study), 9 patients in CP collected at the time of imatinib resistance, 7 in accelerated phase and 11 in BC, In addition 21 healthy donors (10 PB and 11 BM). In 18 patients, genes expression was analyzed during remission as well. Protein expression was investigated by Western Blot and Immunofluorescence. In addition, K562 cells were transfected with Dab plasmids to gain insight about the effects of Dab1/2 on cell proliferation and apoptosis. We found that in CML patients Dab1/2 expression levels were significantly decreased in either BM or PB (p<0.002 and p<0.0004) as compared to healthy subjects. Furthermore, in blast crisis Dab1/2 transcript levels are further decreased. In contrast, at the time of remission, the transcript levels were comparable to normal values. Data analysis of patients included in TOPS studies shows that Dab1 values are lower among those achieving MMR by 12 months (median value 2) compared to those without MMR (median 4,59) although this difference does not appear significant (p=0.15). By analyzing all together the CP patients enrolled in different protocols based on imatinib as front line therapy, this difference became significant (p=0,01). Western Blot and immunofluorescence confirmed the absence of Dab1/2 proteins in course of active CML samples while it reappeared during remission. Dab1 and Dab2 transfection of K562 significantly reduced proliferation (p=0,002). In conclusion, our results show a significant decrease of Dab1/2 expression in blast crisis samples, when compared to CP CML and healthy volunteers, which suggest a role of Dab1/2 in slowing down or suppressing a progression. Among CP CML patients the responders to TKI therapy have been detected to express a lower Dab levels of than non responders. We find those data intriguing and suggesting that molecular factors involved in the regulation of CML progression could be uncoupled from the mechanisms regulating response to TKI therapy. Supported by Novartis Oncology, Clinical Development, TOPS Clinical Correlative Studies Network Disclosures:No relevant conflicts of interest to declare.

The vitamin D transport protein Dab2 is expressed in prostate and colon epithelial cells and modulated by all‐trans‐retinoic acid.

Disabled‐2 (Dab2) is an adapter protein, that along with the membrane receptors megalin and cubilin, is essential for uptake of protein‐complexed 25‐hydroxycholecalciferol (25D3) in the kidney. Previously, we showed that mammary epithelial cells (T‐47D) also express Dab2, megalin, and cubilin and internalize vitamin D‐binding protein (DBP) by receptor‐mediated endocytosis. Moreover, induction of Dab2 and megalin (protein and mRNA) by the differentiating agent all‐trans‐retinoic acid (RA) correlated with increased DBP uptake in mammary cells. A number of other absorptive epithelial tissues also express Dab2, which suggests that these tissues are capable of internalizing the 25D3‐DBP complex. In the present study, we examined the expression of Dab2, megalin, and cubilin in prostate epithelial (PC‐3 and LNCaP) and colon epithelial (Caco‐2) cancer cells and their ability to internalize DBP. Our preliminary results indicate that PC‐3 and Caco‐2 cells express Dab2 (mRNA and protein) and this expression was markedly enhanced when cells were treated with 10 μM RA. Taken together, these are the first studies to our knowledge that have characterized a potential role for Dab2, megalin, and cubilin in the uptake of vitamin D in prostate and colon cells. Supported by NIH 5R03CA128091‐03 and Iowa State University Experiment station to MJR.

Frequent loss of Dab2 protein and infrequent promoter hypermethylation in breast cancer

Disabled-2 (Dab2), a putative tumor suppressor protein, is lost in 80-90% ovarian tumors and ovarian/breast cancer cell lines. The clinical significance of Dab2 protein in breast cancer remains yet unknown. Immunohistochemical analysis of Dab2 protein showed no detectable expression in 67/91 (74%) breast tumors, while all 10 normal tissues showed presence of Dab2 protein. We hypothesized that epigenetic silencing of Dab2 may account for loss of protein in breast cancer. Methylation of Dab2 exon 1, a putative promoter, was analyzed in six breast cancer cell lines and in 54 primary breast tumors by methylation specific PCR. Methylation was observed in MDA-MB-231 and MDA-MB-157 cells and in 6 of 54 (11%) primary breast tumors that also showed loss of Dab2 protein. Expression of Dab2 transcripts was detected in all cell lines except MDA-MB-157. However, none of these six cell lines showed detectable levels of Dab2 protein by western blotting, while non-malignant mammary epithelial cell line MCF 10A showed Dab2 protein expression. To our knowledge this is the first report showing low frequency of Dab2 (putative) promoter methylation (11%) in primary breast tumors. Frequent loss of Dab2 protein (74%) suggest that hypermethylation of Dab2 promoter may only be one of the mechanisms accounting for its loss in breast cancer. Further, in silico analysis of Dab2 3'-UTR revealed existence of miRNA complimentary to this region of the gene, suggesting microRNA mediated targeting of Dab2 mRNA might account for loss of the protein in breast cancer.