The inhibitory role of DOC-2/DAB2 in growth factor receptor-mediated signal cascade. DOC-2/DAB2-mediated inhibition of ERK phosphorylation via binding to Grb2

DOC-2/DAB2 (differentially expressed in ovarian carcinoma-2/disabled 2) appears to be a potential tumor suppressor gene with a growth inhibitory effect on several cancer types. Previously, we have shown that DOC-2/DAB2 suppresses protein kinase C-induced AP-1 activation, which is modulated by serine 24 phosphorylation in the N terminus of DOC-2/DAB2. However, the functional impact of the C terminus of DOC-2/DAB2, containing three proline-rich domains, has not been explored. In this study, we examined this functional role in modulating signaling mediated by peptide growth factor receptor tyrosine kinase, particularly because it involves the interaction with Grb2. Using sequence-specific peptides, we found that the second proline-rich domain of DOC-2/DAB2 is the key binding site to Grb2 in the presence of growth factors. Such elevated binding interrupts the binding between SOS and Grb2, which consequently suppresses downstream ERK phosphorylation. Reduced ERK phosphorylation was restored when the binding between DOC-2/DAB2 and Grb2 was interrupted by a specific peptide or by increasing the expression of Grb2. Furthermore, the C terminus of the DOC-2/DAB2 construct can inhibit the AP-1 activity elicited by growth factors. We conclude that DOC-2/DAB2, a potent negative regulator, can suppress ERK activation by interrupting the binding between Grb2 and SOS that is elicited by peptide growth factors. This study further illustrates that DOC-2/DAB2 has multiple effects on the RAS-mediated signal cascades active in cancer cells.     

Characterization of a novel negative regulator (DOC-2/DAB2) of c-Src in normal prostatic epithelium and cancer

DOC-2/DAB2 is a potent tumor suppressor in many cancer types including prostate cancer. In prostate cancer, expression of DOC-2/DAB2 can inhibit its growth. Our recent studies demonstrate that DOC-2/DAB2 can suppress both protein kinase C and peptide growth factor-elicited signal pathways via the Ras-mitogen-activated protein kinase pathway. In this study, we further showed that the proline-rich domain of DOC-2/DAB2 could also interact with proteins containing the Src homology 3 domain, such as Src and Fgr. The binding of c-Src to DOC-2/DAB2 was enhanced in cells treated with growth factor, and this interaction resulted in c-Src inactivation. The c-Src inactivation was evidenced by the decreased tyrosine 416 phosphorylation of c-Src and reduced downstream effector activation. It appears that DOC-2/DAB2 can bind to Src homology 3 domain of c-Src and maintain it in an inactive conformation. Thus, this study provides a new mechanism for modulating c-Src in prostatic epithelium and cancer.     

The role of DOC-2/DAB2 protein phosphorylation in the inhibition of AP-1 activity. An underlying mechanism of its tumor-suppressive function in prostate cancer.

DOC-2/DAB2, a novel phosphoprotein with signal-transducing capability, inhibits human prostatic cancer cells (Tseng, C.-P., Ely, B. D., Li, Y., Pong, R.-C., and Hsieh, J.-T. (1998) Endocrinology 139, 3542-3553). However, its mechanism of action is not understood completely. This study delineates the functional significance of DOC-2/DAB2 protein phosphorylation and demonstrates that in vivo activation of protein kinase C (PKC) by 12-O-tetradecanoylphorbol-13-acetate (TPA) induces DOC-2/DAB2 phosphorylation, including a serine residue at position 24. Mutation of Ser(24) to Ala reduced DOC-2/DAB2 phosphorylation by PKC. Using a synthetic Ser(24) peptide (APS(24)KKEKKKGSEKTD) or recombinant DOC-2/DAB2 as substrates, PKCbetaII, PKCgamma, and PKCdelta (but not casein kinase II) directly phosphorylated Ser(24) in vitro. This indicates that DOC-2/DAB2 is a PKC-specific substrate. Since expression of wild-type DOC-2/DAB2, but not the S24A mutant, inhibited TPA-induced AP-1 activity in prostatic epithelial cells, phosphorylation of Ser(24) appears to play a critical role in modulating TPA-induced AP-1 activity. Taken together, these data suggest that PKC-regulated phosphorylation of DOC-2/DAB2 protein may help its growth inhibitory function.     

DOC-2/DAB2 is the binding partner of myosin VI

Myosin VI is a molecular motor that moves processively along actin filaments and is believed to play a role in cargo movement in cells. Here we found that DOC-2/DAB2, a signaling molecule inhibiting the Ras cascade, binds to myosin VI at the globular tail domain. DOC-2/DAB2 binds stoichiometrically to myosin VI with one molecule per one myosin VI heavy chain. The C-terminal 122 amino acid residues of DOC-2/DAB2, containing the Grb2 binding site, is identified to be critical for the binding to myosin VI. Actin gliding assay revealed that the binding of DOC-2/DAB2 to myosin VI can support the actin filament gliding by myosin VI, suggesting that it can function as a myosin VI anchoring molecule. The C-terminal domain but not the N-terminal domain of DOC-2/DAB2 functions as a myosin VI anchoring site. The present findings suggest that myosin VI plays a role in transporting DOC-2/DAB2, a Ras cascade signaling molecule, thus involved in Ras signaling pathways.     

Association of the variant rs2243421 of human DOC-2/DAB2 interactive protein gene (hDAB2IP) with gastric cancer in the Chinese Han population

Human DOC-2/DAB2 interactive protein (hDAB2IP) gene is a novel member of the Ras GTPase-activating family and has been demonstrated to be a tumor-suppressor gene that inhibits cell survival and proliferation and induces cell apoptosis. It was reported that the expression level of hDAB2IP in gastric cancer tissue was highly correlated with tumor progression, however, whether hDAB2IP genetic variants are associated with the risk of gastric cancer remains yet unknown. In this case–control study, we conducted a genetic analysis for hDAB2IP variants in 311 patients with gastric cancer and 425 controls from the Chinese Han population. We found that the SNP rs2243421 of hDAB2IP gene with the minor allele C significantly revealed strong association with decreased gastric cancer susceptibility (P = 0.007, adjusted odds ratio [OR] = 0.734, 95%CI = 0.586–0.919). Haplotypes rs2243421 and rs10985332 (HaploType: CC, P = 0.012, aOR = 0.760) and haplotypes rs2243421 and rs555996 (HaploType: CG, P = 0.034, aOR = 0.788) represented the decreased risk of gastric cancer, respectively. On the contrary, rs2243421 and rs555996 showed an elevated susceptibility (HaploType: TG, P = 0.010, aOR = 1.320). Our results for the first time provided new insight into susceptibility factors of hDAB2IP gene variants in carcinogenesis of gastric cancer.     

Cloning of mouse Dab2ip gene, a novel member of the RasGTPase-activating protein family and characterization of its regulatory region in prostate

Disabled homolog 2 (Drosophila) interacting protein (DAB2IP/Dab2IP) is a member of the GTPase-activating protein for downregulating the Ras-mediated signal pathway and TNF-mediated apoptosis. The downregulation of human DAB2IP mRNA levels was detected in prostate cancer cells due to the epigenetic regulation. Here, we isolated a mouse Dab2ip gene with a highly homologous sequence to that of the human and rat gene and mapped it at chromosome 2B. The mDab2ip gene contains 14 exons and 13 introns and spans approximately 65 kb. Exon1 contains at least three splicing variants (Ia, Ib, and Ic). The deduced amino acid sequence of mouse Dab2IP encompasses 1065 residues containing several unique protein interaction motifs as well as a Ras-like GAP-related domain, which shares a high homology with both humans and rats. Data from real-time RT-PCR analysis revealed a diverse expression pattern of the mDab2ip gene in various organs, implying differential regulation of this gene from various tissues. We have mapped a 1.3-kb segment containing a 5'-upstream region from exon Ia as a promoter region (-147/+545) in prostatic epithelial cell lines (TRAMP-C); this region is highly GC-rich, and mDab2ip appears to be a TATA-less promoter. It appears that epigenetic regulation, particularly histone acetylation of the Dab2ip gene promoter, plays an important role in modulating its gene expression in the mouse prostate cancer cell.     

A common genetic variant (97906C>A) of DAB2IP/AIP1 is associated with an increased risk and early onset of lung cancer in Chinese males

DOC-2/DAB2 interactive protein (DAB2IP) is a novel identified tumor suppressor gene that inhibits cell growth and facilitates cell apoptosis. One genetic variant in DAB2IP gene was reported to be associated with an increased risk of aggressive prostate cancer recently. Since DAB2IP involves in the development of lung cancer and low expression of DAB2IP are observed in lung cancer, we hypothesized that the variations in DAB2IP gene can increase the genetic susceptibility to lung cancer. In a case-control study of 1056 lung cancer cases and 1056 sex and age frequency-matched cancer-free controls, we investigated the association between two common polymorphisms in DAB2IP gene (-1420T>G, rs7042542; 97906C>A, rs1571801) and the risk of lung cancer. We found that compared with the 97906CC genotypes, carriers of variant genotypes (97906AC+AA) had a significant increased risk of lung cancer (adjusted odds ratio [OR] = 1.33, 95%CI = 1.04-1.70, P = 0.023) and the number of variant (risk) allele worked in a dose-response manner (P(trend) = 0.0158). Further stratification analysis showed that the risk association was more pronounced in subjects aged less than 60 years old, males, non-smokers, non-drinkers, overweight groups and in those with family cancer history in first or second-degree relatives, and the 97906A interacted with overweight on lung cancer risk. We further found the number of risk alleles (97906A allele) were negatively correlated with early diagnosis age of lung cancer in male patients (P = 0.003). However, no significant association was observed on the -1420T>G polymorphism. Our data suggested that the 97906A variant genotypes are associated with the increased risk and early onset of lung cancer, particularly in males.     

DAB2IP与肿瘤的发生发展

基因DAB2IP（disabled homolog 2-interacting protein,DAB2 interacting protein）,也被称为AIP1（ASK1-interacting protein 1）,其编码的蛋白是Ras GTPase活化蛋白家族[Ras GTPase-activating protein（GAP）]的新成员之一.作为一个肿瘤抑制基因,DAB2IP常在前列腺癌、乳腺癌、肺癌、肝癌等肿瘤中表达下调,其机制与启动子甲基化及Ezh2相关.DAB2IP不仅参与肿瘤的增殖、存活和凋亡过程,还与肿瘤转移密切相关.     

Deletion of SMURF 1 represses ovarian cancer invasion and EMT by modulating the DAB2IP/AKT/Skp2 feedback loop

SMAD ubiquitination regulatory factor 1 (SMURF1) has been described as a tumor suppressor in multiple aggressive cancers. Nevertheless, the potential role of SMURF1 in ovarian cancer invasion and epithelial-to-mesenchymal transition (EMT) remains unclear. The aim of this study was to evaluate the efficacy of SMURF1 on tumor migration and EMT and elucidate the underlying molecular mechanism in ovarian carcinoma. We found elevated SMURF1 in several ovarian cancer cells in both messenger RNA and protein. Additionally, silencing SMURF1 apparently repressed cell proliferation and invasion capacity of SKOV3 and A2780 cells and markedly attenuated expression of linked proteins such as proliferating cellnuclear antigen, matrix metalloproteinase (MMP)-2, and MMP-9. Furthermore, depletion of SMURF1 dramatically impeded EMT progress by modulating EMT biomarkers, with a notable increase in E-cadherin expression accompanied by the decrease in N-cadherin and vimentin in both SKOV3 and A2780 cells. Interestingly, elimination of SMURF1 led to disabled homolog 2 DOC-2/DAB2 interacting protein (DAB2IP) activation and dampened AKT/Skp2 signaling. Most important, depleted of DAB2IP or treatment with the AKT agonist 740Y-P effectively abolished the suppressive effects of SMURF1 knockout on cell invasiveness and EMT process. Taken all data together, these findings demonstrated that the absence of SMURF1 repressed cell proliferation, invasive capability, and EMT process in ovarian cancer through DAB2IP/AKT/Skp2 signaling loops, suggesting that SMURF1 may serve as a new potential therapeutic agent for ovarian cancer.     

Overexpression of DOC-1R Inhibits Cell Cycle G1/S Transition by Repressing CDK2 Expression and Activation

DOC-1R (deleted in oral cancer-1 related) is a novel putative tumor suppressor. This study investigated DOC-1R antitumor activity and the underlying molecular mechanisms. Cell phenotypes were assessed using flow cytometry, BrdU incorporation and CDK2 kinase assays in DOC-1R overexpressing HeLa cells. In addition, RT-PCR and Western blot assays were used to detect underlying molecular changes in these cells. The interaction between DOC-1R and CDK2 proteins was assayed by GST pull-down and immunoprecipitation-Western blot assays. The data showed that DOC-1R overexpression inhibited G1/S phase transition, DNA replication and suppressed CDK2 activity. Molecularly, DOC-1R inhibited CDK2 expression at the mRNA and protein levels, and there were decreased levels of G1-phase cyclins (cyclin D1 and E) and elevated levels of p21, p27, and p53 proteins. Meanwhile, DOC-1R associated with CDK2 and inhibited CDK2 activation by obstructing its association with cyclin E and A. In conclusion, the antitumor effects of DOC-1R may be mediated by negatively regulating G1 phase progression and G1/S transition through inhibiting CDK2 expression and activation.     

Expression patterns of human DAB2IP protein in fetal tissues

Abstract

Human DOC-2/DAB2-interacting protein (DAB2IP) is encoded by a tumor suppressor gene and a newly recognized member of the Ras-GTPase-activating family. DAB2IP is a critical component of many signal transduction pathways mediated by Ras and tumor necrosis factors including apoptosis pathways, and it is involved in the formation of many types of tumors. DAB2IP participates in regulation of gene expression and pluripotency of cells. It has been reported that DAB2IP was expressed in different tumor tissues. Little information is available concerning the expression levels of DAB2IP in normal tissues and cells, however, and no studies of its expression patterns during the development of human embryos have been reported. We examined the expression of DAB2IP during human embryonic development to understand better DAB2IP functions. Human fetuses, weeks 9 to 38, and a newborn were obtained from miscarriages or stillbirths. Tissues were embedded in paraffin to construct arrays that were stained immunohistochemically. The DAB2IP-positive cells were identified and scored based on both the percentage of stained cells and their staining intensities. DAB2IP was expressed in most fetal tissues examined. DAB2IP was expressed primarily in cell cytoplasm throughout the fetal development. The expression levels varied among tissues and different gestational ages. Virtually no expression was observed in the cerebrum, parotid gland, thymus, thyroid gland and spleen. Expression was much greater in the adrenal gland and pancreas; weakly to moderately strong in the endocardium, stomach, kidney, testis and small intestine; and lower in liver, trachea, skin, ovary and endometrium. Its expression in the lung, esophagus and bladder were much weaker to absent.     

人口腔癌症缺失相关蛋白（DOC-1R）的表达、纯化和晶体生长

口腔癌缺失(DeletedinOralCancer-1,DOC-1)基因是近年来被证实的口腔癌中具有抑癌作用的基因。1999年,研究人员通过酵母双杂交实验又发现了与DOC-1相关的另一候选抑癌基因DOC-1R(DOC-1related)。以往的很多实验表明,这两个蛋白无论序列还是功能上都非常相似。然而,其三维结构以及与其他重要蛋白相互作用的机制一直还不清楚,PDB库中也未见其相关同源结构的报道。作者将人DOC-1R基因的cDNA片段克隆至原核表达载体pET-22b( )中,通过IPTG诱导获得高效表达,再经过Ni-NTA亲和层析和Superdex75层析柱纯化,获得了纯度达到96%以上的蛋白。质谱分子量测定显示DOC-1R的分子量为14091.23Da,与理论分子量基本一致;动态光散射实验显示蛋白均一性高达99.0%,可用于晶体生长;采用悬滴气相扩散法筛选,在多个条件下得到了DOC-1R的微晶。为DOC-1R的三维结构解析奠定了坚实的基础。     

Down-regulation of human DAB2IP gene expression mediated by polycomb Ezh2 complex and histone deacetylase in prostate cancer

Human DAB2IP (hDAB2IP), a novel GTPase-activating protein modulating the Ras-mediated signaling and tumor necrosis factor-mediated apoptosis, is a potent growth inhibitor in human prostate cancer (PCa). Loss of hDAB2IP expression in PCa is due to altered epigenetic regulation (i.e. DNA methylation and histone modification) of its promoter region. The elevated polycomb Ezh2, a histone methyltransferase, has been associated with PCa progression. In this study, we have demonstrated that an increased Ezh2 expression in normal prostatic epithelial cells can suppress hDAB2IP gene expression. In contrast, knocking down the endogenous Ezh2 levels in PCa by a specific small interfering RNA can increase hDAB2IP expression. The association of Ezh2 complex (including Eed and Suz12) with hDAB2IP gene promoter is also detected in PCa cells but not in normal prostatic epithelial cells. Increased Ezh2 expression in normal prostatic epithelial cells by cDNA transfection facilitates the recruitment of other components of Ezh2 complex to the hDAB2IP promoter region accompanied with the increased levels of methyl histone H3 (H3) and histone deacetylase (HDAC1). Consistently, data from PCa cells transfected with Ezh2 small interfering RNA demonstrated that reduced Ezh2 levels resulted in the dissociation of Ezh2 complex accompanied with decreased levels of both methyl H3 and HDAC1 from hDAB2IP gene promoter. We further unveiled that the methylation status of Lys-27 but not Lys-9 of H3 in hDAB2IP promoter region is consistent with the hDAB2IP levels in both normal prostatic epithelial cells and PCa cells. Together, we conclude that hDAB2IP gene is a target gene of Ezh2 in prostatic epithelium, which provides an underlying mechanism of the down-regulation of hDAB2IP gene in PCa.     

Expression patterns of human DAB2IP protein in fetal tissues

Human DOC-2/DAB2-interacting protein (DAB2IP) is encoded by a tumor suppressor gene and a newly recognized member of the Ras-GTPase-activating family. DAB2IP is a critical component of many signal transduction pathways mediated by Ras and tumor necrosis factors including apoptosis pathways, and it is involved in the formation of many types of tumors. DAB2IP participates in regulation of gene expression and pluripotency of cells. It has been reported that DAB2IP was expressed in different tumor tissues. Little information is available concerning the expression levels of DAB2IP in normal tissues and cells, however, and no studies of its expression patterns during the development of human embryos have been reported. We examined the expression of DAB2IP during human embryonic development to understand better DAB2IP functions. Human fetuses, weeks 9 to 38, and a newborn were obtained from miscarriages or stillbirths. Tissues were embedded in paraffin to construct arrays that were stained immunohistochemically. The DAB2IP-positive cells were identified and scored based on both the percentage of stained cells and their staining intensities. DAB2IP was expressed in most fetal tissues examined. DAB2IP was expressed primarily in cell cytoplasm throughout the fetal development. The expression levels varied among tissues and different gestational ages. Virtually no expression was observed in the cerebrum, parotid gland, thymus, thyroid gland and spleen. Expression was much greater in the adrenal gland and pancreas; weakly to moderately strong in the endocardium, stomach, kidney, testis and small intestine; and lower in liver, trachea, skin, ovary and endometrium. Its expression in the lung, esophagus and bladder were much weaker to absent.     

miR-145靶向调控DAB2对前列腺癌PC3细胞迁移和侵袭能力的影响

已有研究表明, miR-145在多种肿瘤中低表达, 并与细胞增殖和转移相关。文章通过生物信息学分析并结合体外实验鉴定, 发现DAB2(Disabled homolog 2)为miR-145在肿瘤转移过程中累及的新靶点。DAB2一直被认为是一个重要的抑癌基因, 在多种肿瘤标本中表达低下。然而, 研究发现, 在具高侵袭能力的前列腺癌细胞株PC3中DAB2基因却呈较高水平表达。另外, 外源表达miR-145能显著下调 DAB2表达水平, 并抑制PC3细胞的迁移和侵袭能力, 且这种miR-145诱导的PC3细胞功能缺陷能被DAB2过表达修复。上述结果表明, miR-145能通过靶向调控DAB2而影响高侵袭前列腺癌细胞的迁移和侵袭能力。     

p12(DOC-1), a growth suppressor, associates with DNA polymerase alpha/primase.

p12(DOC-1) is a growth suppressor identified and isolated from normal keratinocytes. Ectopic expression of p12(DOC-1) in squamous carcinoma cells led to the reversion of in vitro transformation phenotypes including anchorage independence, doubling time, and morphology. Here we report that p12(DOC-1) associates with DNA polymerase alpha/primase (pol-alpha:primase) in vitro and in cells. The pol-alpha:primase binding domain in p12(DOC-1) is mapped to the amino-terminal six amino acid (MSYKPN). The biological effect of p12(DOC-1) on pol-alpha:primase was examined using in vitro DNA replication assays. Using the SV40 DNA replication assay, p12(DOC-1) suppresses DNA replication, leveling at approximately 50%. Similar results were obtained using the M13 single-stranded DNA synthesis assay. Analysis of the DNA replication products revealed that p12(DOC-1) affects the initiation step, not the elongation phase. The p12(DOC-1) suppression of DNA replication is likely to be mediated either by a direct inhibitory effect on pol-alpha:primase or by its effect on cyclin-dependent kinase 2 (CDK2), a recently identified p12(DOC-1)-associated protein known to stimulate DNA replication by phosphorylating pol-alpha:primase. p12(DOC-1) suppresses CDK2-mediated phosphorylation of pol-alpha:primase. These data support a role of p12(DOC-1) as a regulator of DNA replication by direct inhibition of pol-alpha:primase or by negatively regulating the CDK2-mediated phosphorylation of pol-alpha:primase.