The rac activator Tiam1 is a Wnt-responsive gene that modifies intestinal tumor development

Mutations in the canonical Wnt signaling pathway leading to its activation are known to cause the majority of intestinal tumors. However, few genes targeted by this pathway have been demonstrated to affect tumor development in vivo. Here we show that Tiam1, a selective Rac GTPase activator, is a Wnt-responsive gene expressed in the base of intestinal crypts and up-regulated in mouse intestinal tumors and human colon adenomas. Moreover, by comparing tumor development in APC mutant Min (multiple intestinal neoplasia) mice expressing or lacking Tiam1, we found that Tiam1 deficiency significantly reduces the formation and growth of polyps in vivo. However, invasion of malignant intestinal tumors is enhanced by a lack of Tiam1. In line with this, knock-down of Tiam1 reduced the growth potential of human colorectal cancer cells and their ability to form E-cadherin-based adhesions, a prerequisite for local invasion of tumor cells. Our data indicate a novel cross-talk between Tiam1-Rac and canonical Wnt-signaling pathways that influences intestinal tumor formation and progression.     

Drosophila APC2 and APC1 play overlapping roles in wingless signaling in the embryo and imaginal discs

The regulation of signal transduction plays a key role in cell fate choices, and its disregulation contributes to oncogenesis. This duality is exemplified by the tumor suppressor APC. Originally identified for its role in colon tumors, APC family members were subsequently shown to negatively regulate Wnt signaling in both development and disease. The analysis of the normal roles of APC proteins is complicated by the presence of two APC family members in flies and mice. Previous work demonstrated that, in some tissues, single mutations in each gene have no effect, raising the question of whether there is functional overlap between the two APCs or whether APC-independent mechanisms of Wnt regulation exist. We addressed this by eliminating the function of both Drosophila APC genes simultaneously. We find that APC1 and APC2 play overlapping roles in regulating Wingless signaling in the embryonic epidermis and the imaginal discs. Surprisingly, APC1 function in embryos occurs at levels of expression nearly too low to detect. Further, the overlapping functions exist despite striking differences in the intracellular localization of the two APC family members.     

Loss of transgelin in repeated bouts of ulcerative colitis-induced colon carcinogenesis

Though ulcerative colitis (UC)-associated colon cancer develops from dysplastic lesions caused by chronic inflammation, the specific mechanistic link between chronic inflammation and carcinogenesis in colon has not been integrated into molecular understanding. We therefore established an experimental animal model for colitic cancer, and used proteomic analysis, based on 2-DE and MALDI-TOF MS, to identify proteins involved in colitic cancer. In our model, 6-week-old C57BL/6J mice were exposed to 15 cycles of dextran sodium sulfate (DSS), with each cycle consisting of 0.7% DSS for 1 week followed by distilled water for 10 days. Colorectal tumors developed in 22 of 24 mice (91.6%), with a tumor multiplicity of 1.727 per tumor-bearing mouse. Comparative 2-DE analysis showed that 38 protein spots were differentially expressed in colon tumors and normal colon. We identified 27 of these proteins, including GRP94, HSC70, enolase, prohibitin, and transgelin. The reduction of transgelin expression in mouse colon tumors was confirmed by Western blotting and immunohistochemistry. We also found that transgelin expression was significantly reduced in human colon tumors compared with adjacent nontumorous tissues. In conclusion, these results suggest that loss of transgelin could be a candidate for biomarker of repeated colitis-associated colon cancer.     

Mucin depleted foci, colonic preneoplastic lesions lacking Muc2, show up-regulation of Tlr2 but not bacterial infiltration

Mucin depleted foci (MDF) are precancerous lesions of the colon in carcinogen-treated rodents and humans at high risk. Since MDF show signs of inflammation we hypothesized that the defective mucous production would expose them to the risk of being penetrated by intestinal bacteria, which can be sensed by Toll-like receptors (Tlrs) and activate inflammatory pathways. To verify this hypothesis we tested the expression of 84 genes coding for Tlrs and associated pathways using RT-qPCR in MDF (n = 7) from 1,2-dimethylhydrazine (DMH)-treated rats. Among the 84 tested genes, 26 were differentially expressed in MDF with 5 genes significantly up-regulated and 21 down-regulated when compared to the normal mucosa. Tlr2, as well as other downstream genes (Map4k4, Hspd1, Irak1, Ube2n), was significantly up-regulated. Among the genes regulating the NFkB pathway, only Map4k4 was significantly up-regulated, while 19 genes were not varied and 6 were down-regulated. Tlr2 protein was weakly expressed both in normal mucosa and MDF. To determine whether inflammation observed in MDF could be caused by bacteria contacting or infiltrating crypts, we performed fluorescence in situ hybridization (FISH) experiments with a rRNA universal bacterial probe. None of the 21 MDF tested, showed bacteria inside the crypts, while among the colonic tumors (n = 15), only one had very few bacteria on the surface and on the surrounding normal mucosa. In conclusion, the up-regulation of Tlr2 in MDF, suggests a link between this receptor and carcinogenesis, possibly related to a defective barrier function of these lesions. The data of FISH experiments do not support the hypothesis that inflammation in MDF and tumors is stimulated by bacterial infiltration.     

Ikaros isoform x is selectively expressed in myeloid differentiation

The Ikaros gene is alternately spliced to generate multiple DNA-binding and nonbinding isoforms that have been implicated as regulators of hematopoiesis, particularly in the lymphoid lineages. Although early reports of Ikaros mutant mice focused on lymphoid defects, these mice also show significant myeloid, erythroid, and stem cell defects. However, the specific Ikaros proteins expressed in these cells have not been determined. We recently described Ikaros-x (Ikx), a new Ikaros isoform that is the predominant Ikaros protein in normal human hematopoietic cells. In this study, we report that the Ikx protein is selectively expressed in human myeloid lineage cells, while Ik1 predominates in the lymphoid and erythroid lineages. Both Ik1 and Ikx proteins are expressed in early human hematopoietic cells (Lin(-)CD34(+)). Under culture conditions that promote specific lineage differentiation, Ikx is up-regulated during myeloid differentiation but down-regulated during lymphoid differentiation from human Lin(-)CD34(+) cells. We show that Ikx and other novel Ikaros splice variants identified in human studies are also expressed in murine bone marrow. In mice, as in humans, the Ikx protein is selectively expressed in the myeloid lineage. Our studies suggest that Ikaros proteins function in myeloid, as well as lymphoid, differentiation and that specific Ikaros isoforms may play a role in regulating lineage commitment decisions in mice and humans.     

结肠癌中核内miRNA的激活调控作用研究

为了探究增强子介导的核内miRNA在结肠癌发生中的作用,本研究筛选了结肠癌中的差异表达的miRNA数据、结肠的特异性增强子数据、结肠癌中差异表达基因数据,利用细胞核内miRNA靶向增强子预测算法,筛选miRNA调控的结肠特异性增强子;利用增强子靶基因预测数据,筛选核内miRNA调控的差异表达靶基因,并且构建核内miRNA-靶基因网络,并通过网络的分析和筛选获得结肠癌中关键的致病基因,同时对网络中的靶基因进行GO的功能注释。结果表明,我们所构建的核内miRNA-激活调控靶基因网络包含miRNA-靶基因关系对2 121个,259个节点,其中包含34个下调基因、183个上调的基因,7个下调的miRNA,35个上调的miRNA。而后我们分析了网络进行的节点度的整体分布情况,发现网络中大部分的节点的度都是小于10的,仅有少量miRNA结合和部分的差异表达基因节点的度大于10。核内miRNA主要通过激活调控了一些应激反应相关的功能和,同时,抑制调控了细胞周期、细胞凋亡、细胞死亡巨噬细胞代谢等相关功能,通过激活和抑制相关功能诱发结肠癌的发生。从核内miRNA的激活调控角度研究结肠癌的发病机制,是对原有细胞浆中miRNA抑制调控机制的补充,也为结肠癌的系统研究提供了新的视野。     

MUC1/sec-expressing tumors are rejected in vivo by a T cell-dependent mechanism and secrete high levels of CCL2

MUC1/sec is a secreted form of the glycoprotein mucin 1 (MUC1). To characterize the role that MUC1 and MUC1/sec have in tumor progression, these genes were expressed in DA-3 mammary tumor cells. DA-3 cells and DA-3 cells expressing the transmembrane MUC1 gene (DA-3/TM) grow with similar kinetics in BALB/c mice. Surprisingly, DA-3 cells expressing and secreting MUC1/sec (DA-3/sec) fail to form tumors in vivo. The mechanism of rejection was evaluated using mice deficient in constituents of the immune system. All mice lacking IFN-gamma, NK, NKT, or macrophages formed DA-3/sec tumors that regressed shortly after implantation. However, progressively growing DA-3/sec tumors developed in mice devoid of T lymphocytes. The importance of T lymphocytes in the rejection of DA-3/sec tumors was further supported by detection of DA-3-specific CTL in mice challenged with the DA-3/sec tumor. Recruitment of appropriate APC and effector cells is an important first step in the tumor clearance. Indeed, DA-3/sec cells or cell supernatants recruited 3-4 times as many macrophages as DA-3/TM cells in vivo, suggesting that a secreted chemotactic product is produced from DA-3/sec cells. RNA and protein analysis of DA-3/sec cells revealed that several genes are up-regulated by MUC1/sec expression, including MCP-1 (CCL-2). These results suggest DA-3/sec cells are capable of recruiting immune cells, and that rejection of DA-3/sec tumors, although aided by cells of the innate immune response, is ultimately due to T cell-mediated events.     

Identification of differentially expressed microRNAs and their PKC-isoform specific gene network prediction during hypoxic pre-conditioning and focal cerebral ischemia of mice

We previously reported the involvement of conventional protein kinase C (cPKC) βII, γ, novel PKC (nPKC) ε and their interacting proteins in hypoxic pre-conditioning (HPC)-induced neuroprotection. In this study, the large-scale miRNA microarrays and bioinformatics analysis were used to determine the differentially expressed miRNAs and their PKC-isoform specific gene network in mouse brain after HPC and 6?h middle cerebral artery occlusion (MCAO). We found 4 up-regulated and 13 down-regulated miRNAs in the cortex of HPC mice, 26 increased and 39 decreased gene expressions of miRNAs in the peri-infarct region of 6?h MCAO mice, and 11 up-regulated and 22 down-regulated miRNAs in the peri-infarct region of HPC and 6?h MCAO mice. Based on Diff Score, 19 differentially expressed miRNAs were identified in HPC and 6?h MCAO mouse brain. Then the miRNA-gene-network of 19 specified miRNAs target genes of cPKCβII, γ and nPKCε-interacting protein was predicted by using bioinformatics analysis of genome databases. Furthermore, the down-regulated miR-615-3p during HPC had a detrimental effect on the oxygen-glucose deprivation (OGD)-induced N2A cell injury. These results suggested that the identified 19 miRNAs, notably miR-615-3p, might target these genes of cPKCβII, γ and nPKCε-interacting proteins involved in HPC-induced neuroprotection.     

A Proteomics Analysis Reveals 9 Up-Regulated Proteins Associated with Altered Cell Signaling in Colon Cancer Patients

Colorectal cancer is the second most common cancer in women and third most common cancer in men. Cell signaling alterations in colon cancer, especially in aggressive metastatic tumors, require further investigations. The present study aims to compare the expression pattern of proteins associated with cell signaling in paired tumor and non-tumor samples of patients with colon cancer, as well as to define the cluster of proteins to differentiate patients with non-metastatic (Dukes’ grade B) and metastatic (Dukes’ grade C&D) colon cancer. Frozen tumor and non-tumor samples were collected after tumor resection from 19 patients with colon cancer. The Panorama? Antibody Microarray-Cell Signaling kits were used for the analyses. The expression ratios of paired tumor/non-tumor samples were calculated for the each protein. We employed R packages ‘samr’, ‘gplots’, ‘supclust’ (pelora, wilma algorithms), ‘glmnet’ for the differential expression analysis, supervised clustering and penalized logistic regression. Significance analysis of microarrays revealed 9 significantly up-regulated proteins, including protein kinase C gamma, c-Myc, MDM2, pan cytokeratin, and 1 significantly down-regulated protein (GAP1) in tumoral mucosa. Pan-cytokeratin and APP were up-regulated in tumor versus non-tumor tissue, and were selected in the predictive cluster to discriminate colon cancer type. Higher levels of S-100b and phospho-Tau-pSer199/202 were confirmed as the predictors of non-metastatic colon cancer by all employed regression/clustering methods. Deregulated proteins in colon cancer are involved in oncogenic signal transduction, cell cycle control, and regulation of cytoskeleton/transport. Further studies are needed to validate potential protein markers of colon cancer development and metastatic progression.     

Keratin 18 attenuates estrogen receptor α-mediated signaling by sequestering LRP16 in cytoplasm

Background

As a key player in suppression of colon tumorigenesis, Adenomatous Polyposis Coli (APC) has been widely studied to determine its cellular functions. However, inconsistencies of commercially available APC antibodies have limited the exploration of APC function. APC is implicated in spindle formation by direct interactions with tubulin and microtubule-binding protein EB1. APC also interacts with the actin cytoskeleton to regulate cell polarity. Until now, interaction of APC with the third cytoskeletal element, intermediate filaments, has remained unexamined.

Results

We generated an APC antibody (APC-M2 pAb) raised against the 15 amino acid repeat region, and verified its reliability in applications including immunoprecipitation, immunoblotting, and immunofluorescence in cultured cells and tissue. Utilizing this APC-M2 pAb, we immunoprecipitated endogenous APC and its binding proteins from colon epithelial cells expressing wild-type APC. Using Liquid Chromatography Tandem Mass Spectrometry (LC-MS/MS), we identified 42 proteins in complex with APC, including β-catenin and intermediate filament (IF) proteins lamin B1 and keratin 81. Association of lamin B1 with APC in cultured cells and human colonic tissue was verified by co-immunoprecipitation and colocalization. APC also colocalized with keratins and remained associated with IF proteins throughout a sequential extraction procedure.

Conclusion

We introduce a versatile APC antibody that is useful for cell/tissue immunostaining, immunoblotting and immunoprecipitation. We also present evidence for interactions between APC and IFs, independent of actin filaments and microtubules. Our results suggest that APC associates with all three major components of the cytoskeleton, thus expanding potential roles for APC in the regulation of cytoskeletal integrity.     

Chronic Ethanol Exposure Produces Time- and Brain Region-Dependent Changes in Gene Coexpression Networks

Repeated ethanol exposure and withdrawal in mice increases voluntary drinking and represents an animal model of physical dependence. We examined time- and brain region-dependent changes in gene coexpression networks in amygdala (AMY), nucleus accumbens (NAC), prefrontal cortex (PFC), and liver after four weekly cycles of chronic intermittent ethanol (CIE) vapor exposure in C57BL/6J mice. Microarrays were used to compare gene expression profiles at 0-, 8-, and 120-hours following the last ethanol exposure. Each brain region exhibited a large number of differentially expressed genes (2,000-3,000) at the 0- and 8-hour time points, but fewer changes were detected at the 120-hour time point (400-600). Within each region, there was little gene overlap across time (~20%). All brain regions were significantly enriched with differentially expressed immune-related genes at the 8-hour time point. Weighted gene correlation network analysis identified modules that were highly enriched with differentially expressed genes at the 0- and 8-hour time points with virtually no enrichment at 120 hours. Modules enriched for both ethanol-responsive and cell-specific genes were identified in each brain region. These results indicate that chronic alcohol exposure causes global ‘rewiring‘ of coexpression systems involving glial and immune signaling as well as neuronal genes.     

Strategic compartmentalization of Toll-like receptor 4 in the mouse gut

Pattern recognition receptors (PRRs), which include the Toll-like receptors (TLRs), are involved in the innate immune response to infection. TLR4 is a model for the TLR family and is the main LPS receptor. We wanted to determine the expression of TLR4 and compare it with that of TLR2 and CD14 along the gastrointestinal mucosa of normal and colitic BALB/c mice. Colitis was induced with 2.5% dextran sodium sulfate (DSS). Mucosa from seven segments of the digestive tract (stomach, small intestine in three parts, and colon in three parts) was isolated by two different methods. Mucosal TLR4, CD14, TLR2, MyD88, and IL-1beta mRNA were semiquantified by Northern blotting. TLR4 protein was determined by Western blotting. TLR4/MD-2 complex and CD14 were evaluated by immunohistochemistry. PRR genes were constitutively expressed and were especially stronger in colon. TLR4 and CD14 mRNA were increased in the distal colon, but TLR2 mRNA was expressed more strongly in the proximal colon, and MyD88 had a uniform expression throughout the gut. Accordingly, TLR4 and CD14 protein levels were higher in the distal colon. TLR4/MD-2 and CD14 were localized at crypt bottom epithelial cells. TLR4/MD2, but not CD14, was found in mucosal mononuclear cells. Finally, DSS-induced inflammation was localized in the distal colon. All genes studied were up-regulated during DSS-induced inflammation, but the normal colon-stressed gut distribution was preserved. Our findings demonstrate that TLR4, CD14, and TLR2 are expressed in a compartmentalized manner in the mouse gut and provide novel information about the in vivo localization of PRRs.     

Time-course microarray analysis for identifying candidate genes involved in obesity-associated pathological changes in the mouse colon

Background

Obesity is known to increase the risk of colorectal cancer. However, mechanisms underlying the pathogenesis of obesity-induced colorectal cancer are not completely understood. The purposes of this study were to identify differentially expressed genes in the colon of mice with diet-induced obesity and to select candidate genes as early markers of obesity-associated abnormal cell growth in the colon.

Methods

C57BL/6N mice were fed normal diet (11% fat energy) or high-fat diet (40% fat energy) and were euthanized at different time points. Genome-wide expression profiles of the colon were determined at 2, 4, 8, and 12 weeks. Cluster analysis was performed using expression data of genes showing log₂ fold change of ≥1 or ≤?1 (twofold change), based on time-dependent expression patterns, followed by virtual network analysis.

Results

High-fat diet-fed mice showed significant increase in body weight and total visceral fat weight over 12 weeks. Time-course microarray analysis showed that 50, 47, 36, and 411 genes were differentially expressed at 2, 4, 8, and 12 weeks, respectively. Ten cluster profiles representing distinguishable patterns of genes differentially expressed over time were determined. Cluster 4, which consisted of genes showing the most significant alterations in expression in response to high-fat diet over 12 weeks, included Apoa4 (apolipoprotein A-IV), Ppap2b (phosphatidic acid phosphatase type 2B), Cel (carboxyl ester lipase), and Clps (colipase, pancreatic), which interacted strongly with surrounding genes associated with colorectal cancer or obesity.

Conclusions

Our data indicate that Apoa4, Ppap2b, Cel, and Clps are candidate early marker genes associated with obesity-related pathological changes in the colon. Genome-wide analyses performed in the present study provide new insights on selecting novel genes that may be associated with the development of diseases of the colon.