Etk/Bmx Regulates Proteinase-Activated-Receptor1 (PAR1) in Breast Cancer Invasion: Signaling Partners,Hierarchy and Physiological Significance

Background

While protease-activated-receptor 1 (PAR₁) plays a central role in tumor progression, little is known about the cell signaling involved.

Methodology/Principal Findings

We show here the impact of PAR₁ cellular activities using both an orthotopic mouse mammary xenograft and a colorectal-liver metastasis model in vivo, with biochemical analyses in vitro. Large and highly vascularized tumors were generated by cells over-expressing wt hPar1, Y397Z hPar1, with persistent signaling, or Y381A hPar1 mutant constructs. In contrast, cells over-expressing the truncated form of hPar1, which lacks the cytoplasmic tail, developed small or no tumors, similar to cells expressing empty vector or control untreated cells. Antibody array membranes revealed essential hPar1 partners including Etk/Bmx and Shc. PAR₁ activation induces Etk/Bmx and Shc binding to the receptor C-tail to form a complex. Y/A mutations in the PAR₁ C-tail did not prevent Shc-PAR₁ association, but enhanced the number of liver metastases compared with the already increased metastases obtained with wt hPar1. We found that Etk/Bmx first binds via the PH domain to a region of seven residues, located between C378-S384 in PAR₁ C-tail, enabling subsequent Shc association. Importantly, expression of the hPar1-7A mutant form (substituted A, residues 378-384), which is incapable of binding Etk/Bmx, resulted in inhibition of invasion through Matrigel-coated membranes. Similarly, knocking down Etk/Bmx inhibited PAR₁-induced MDA-MB-435 cell migration. In addition, intact spheroid morphogenesis of MCF10A cells is markedly disrupted by the ectopic expression of wt hPar1. In contrast, the forced expression of the hPar1-7A mutant results in normal ball-shaped spheroids. Thus, by preventing binding of Etk/Bmx to PAR₁ -C-tail, hPar1 oncogenic properties are abrogated.

Conclusions/Significance

This is the first demonstration that a cytoplasmic portion of the PAR₁ C-tail functions as a scaffold site. We identify here essential signaling partners, determine the hierarchy of binding and provide a platform for therapeutic vehicles via definition of the critical PAR₁ -associating region in the breast cancer signaling niche.     

Survival of the Fittest: Positive Selection of CD4+ T Cells Expressing a Membrane-Bound Fusion Inhibitor Following HIV-1 Infection

Although a variety of genetic strategies have been developed to inhibit HIV replication, few direct comparisons of the efficacy of these inhibitors have been carried out. Moreover, most studies have not examined whether genetic inhibitors are able to induce a survival advantage that results in an expansion of genetically-modified cells following HIV infection. We evaluated the efficacy of three leading genetic strategies to inhibit HIV replication: 1) an HIV-1 tat/rev-specific small hairpin (sh) RNA; 2) an RNA antisense gene specific for the HIV-1 envelope; and 3) a viral entry inhibitor, maC46. In stably transduced cell lines selected such that >95% of cells expressed the genetic inhibitor, the RNA antisense envelope and viral entry inhibitor maC46 provided the strongest inhibition of HIV-1 replication. However, when mixed populations of transduced and untransduced cells were challenged with HIV-1, the maC46 fusion inhibitor resulted in highly efficient positive selection of transduced cells, an effect that was evident even in mixed populations containing as few as 1% maC46-expressing cells. The selective advantage of the maC46 fusion inhibitor was also observed in HIV-1-infected cultures of primary T lymphocytes as well as in HIV-1-infected humanized mice. These results demonstrate robust inhibition of HIV replication with the fusion inhibitor maC46 and the antisense Env inhibitor, and importantly, a survival advantage of cells expressing the maC46 fusion inhibitor both in vitro and in vivo. Evaluation of the ability of genetic inhibitors of HIV-1 replication to confer a survival advantage on genetically-modified cells provides unique information not provided by standard techniques that may be important in the in vivo efficacy of these genes.     

Surface-Based Analysis on Shape and Fractional Anisotropy of White Matter Tracts in Alzheimer's Disease

Background

White matter disruption has been suggested as one of anatomical features associated with Alzheimer''s disease (AD). Diffusion tensor imaging (DTI), which has been widely used in AD studies, obtains new insights into the white matter structure.

Methods

We introduced surface-based geometric models of the deep white matter tracts extracted from DTI, allowing the characterization of their shape variations relative to an atlas as well as fractional anisotropy (FA) variations on the atlas surface through large deformation diffeomorphic metric mapping (LDDMM). We applied it to assess local shapes and FA variations of twenty-three deep white matter tracts in 13 patients with AD and 19 healthy control subjects.

Results

Our results showed regionally-specific shape abnormalities and FA reduction in the cingulum tract and the sagittal stratum tract in AD, suggesting that disruption in the white matter tracts near the temporal lobe may represent the secondary consequence of the medial temporal lobe pathology in AD. Moreover, the regionally-specific patterns of FA and shape of the white matter tracts were shown to be of sufficient sensitivity to robustly differentiate patients with AD from healthy comparison controls when compared with the mean FA and volumes within the regions of the white matter tracts. Finally, greater FA or deformation abnormalities of the white matter tracts were associated with lower MMSE scores.

Conclusion

The regionally-specific shape and FA patterns could be potential imaging markers for differentiating AD from normal aging.     

TRIM22 inhibits the TRAF6-stimulated NF-κB pathway by targeting TAB2 for degradation

Tripartite motif containing 22 (TRIM22), a member of the TRIM/RBCC family, has been reported to activate the nuclear factor-kappa B (NF-κB) pathway in unstimulated macrophage cell lines, but the detailed mechanisms governing this activation remains unclear. We investigated this mechanism in HEK293T cells. We found that overexpression of TRIM22 could activate the NF-κB pathway and conversely, could inhibit the tumor necrosis factor receptor-associated factor 6 (TRAF6)-stimulated NF-κB pathway in HEK293T cells. Further experiments showed that TRIM22 could decrease the self-ubiquitination of TRAF6, and interact with and degrade transforming growth factor-β activated kinase 1 binding protein 2 (TAB2), and that these effects could be partially rescued by a TRIM22 RING domain deletion mutant. Collectively, our data indicate that overexpression of TRIM22 may negatively regulate the TRAF6-stimulated NF-κB pathway by interacting with and degrading TAB2.     

Affinity maturation of anti-TNF-alpha scFv with somatic hypermutation in non-B cells

Activation-induced cytidine deaminase (AID) is required for the generation of antibody diversity through initiating both somatic hypermutation (SHM) and class switch recombination. A few research groups have successfully used the feature of AID for generating mutant libraries in directed evolution of target proteins in B cells in vitro. B cells, cultured in suspension, are not convenient for transfection and cloning. In this study, we established an AID-based mutant accumulation and sorting system in adherent human cells. Mouse AID gene was first transfected into the human non-small cell lung carcinoma H1299 cells, and a stable cell clone (H1299-AID) was selected. Afterwards, anti-hTNF-αscFv (ATscFv) was transfected into H1299-AID cells and ATscFv was displayed on the surface of H1299-AID cells. By 4-round amplification/flow cytometric sorting for cells with the highest affinities to hTNF-alpha, two ATscFv mutant gene clones were isolated. Compared with the wild type ATscFv, the two mutants were much more efficient in neutralizing cytotoxicity of hTNF-alpha. The results indicate that directed evolution by somatic hypermutation can be carried out in adherent non-B cells, which makes directed evolution in mammalian cells easier and more efficient.     

HaCaT细胞中FUT4表达与其启动子区甲基化的相关性分析

岩藻糖基转移酶Ⅳ(Fucosyltransferase Ⅳ,FUT4)在正常细胞中表达量很低,但其低表达的调控机制以及是否受其启动子甲基化调控并不十分清楚。文章采用Western blot、免疫荧光和Real-time PCR的方法检测正常人永生化表皮细胞系HaCaT细胞FUT4的表达,观察DNA甲基转移酶抑制剂5-aza-dC处理对FUT4表达的影响。应用甲基化特异性PCR方法分析HaCaT细胞中FUT4启动子甲基化状态。结果表明,HaCaT细胞中FUT4的表达水平明显低于人表皮鳞癌细胞A431和SCC12。5 μmol/L的5-aza-dC处理72 h的HaCaT细胞,其FUT4 mRNA水平明显升高,并且与未经5-aza-dC处理的对照组相比,U引物扩增检测到的产物量增加,M 引物扩增检测到的产物量明显减少。这些结果表明,HaCaT细胞中FUT4的低表达可能与其启动子区CpG岛甲基化有关。     

FUT4-siRNA增强5-aza-dC对鳞癌细胞增殖和迁移的抑制

岩藻糖基转移酶IV（fucosyltransferase IV,FUT4）是催化蛋白质岩藻糖基化的关键酶．已经证明,FUT4-siRNA能够抑制鳞癌细胞的增殖．5-氮杂-2-脱氧胞苷（5-aza-dC）是临床常用化疗药物,但5-aza-dC是否对鳞癌有治疗作用,以及与FUT4-siRNA联合使用能否加强对鳞癌细胞增殖和迁移的抑制尚不清楚．本研究以鳞癌细胞系A431和SCC12为对象,探讨5-aza-dC及其与FUT4-siRNA联合使用对细胞增殖和迁移的影响．MTT结合流式细胞周期分析显示,5-aza-dC处理A431和SCC12细胞4 d后,细胞增殖被明显抑制,抑制率分别为18%和20% (P<0.05);与对照组比较,加药处理组G1期细胞数量减少,S期细胞数量明显增加．Western印迹结果揭示,A431细胞FUT4表达水平较SCC12细胞高．经5-aza-dC处理后SCC12细胞FUT4表达有所增加,但仍低于A431细胞中的表达．FUT4-siRNA转染结合台盼蓝活细胞记数证明,FUT4-siRNA明显降低细胞FUT4表达,5-aza-dC处理同时转染FUT4-siRNA的A431和SCC12细胞增殖进一步被抑制,抑制率分别为54%和60% (P<0.05)．细胞划痕法显示,5-aza-dC与FUT4-siRNA联合使用,对细胞迁移能力的抑制作用比5-aza-dC单独使用增强．上述结果提示,5-aza-dC通过诱导细胞S期阻滞抑制鳞癌细胞增殖,FUT4-siRNA与5-aza+dC联合使用可加强对细胞增殖和迁移的抑制．