PGD2 plays roles in allergic inflammation via specific receptors, the PGD receptor designated DP and CRTH2 (chemoattractant receptor homologous molecule expressed on Th2 cells). We generated mutant mice carrying a targeted disruption of the CRTH2 gene to investigate the functional roles of CRTH2 in cutaneous inflammatory responses. CRTH2-deficent mice were fertile and grew normally. Ear-swelling responses induced by hapten-specific IgE were less pronounced in mutant mice, giving 35–55% of the responses of normal mice. Similar results were seen in mice treated with a hemopoietic PGD synthase inhibitor, HQL-79, or a CRTH2 antagonist, ramatroban. The reduction in cutaneous responses was associated with decreased infiltration of lymphocytes, eosinophils, and basophils and decreased production of macrophage-derived chemokine and RANTES at inflammatory sites. In models of chronic contact hypersensitivity induced by repeated hapten application, CRTH2 deficiency resulted in a reduction by approximately half of skin responses and low levels (63% of control) of serum IgE production, although in vivo migration of Langerhans cells and dendritic cells to regional lymph nodes was not impaired in CRTH2-deficient mice. In contrast, delayed-type hypersensitivity to SRBC and irritation dermatitis in mutant mice were the same as in wild-type mice. These findings indicate that the PGD2-CRTH2 system plays a significant role in chronic allergic skin inflammation. CRTH2 may represent a novel therapeutic target for treatment of human allergic disorders, including atopic dermatitis.

The tumor suppressor p21 acts as a cell cycle inhibitor and has also been shown to regulate gene expression by functioning as a transcription corepressor. Here, we identified p21-regulated microRNAs (miRNAs) by sequencing small RNAs from isogenic p21؉ MicroRNAs (miRNAs) are small noncoding RNAs (ϳ22 nucleotides [nt]) that function in diverse biological processes, including cellular proliferation, apoptosis, and differentiation (1-4). miRNAs promote mRNA decay and/or reduce translation by base pairing to partially complementary sequences in the 3= untranslated region (UTR) of target mRNAs (3,(5)(6)(7)(8)(9). Altered miRNA expression is frequent in human cancers. Dysregulation of miRNAs in cancer may occur via several mechanisms, including genetic or epigenetic alterations, defects in miRNA processing, or deregulation of miRNA-regulating transcription factors (10). miRNAs, including let-7, miR-34a, and miR-16 (11-15), act as tumor suppressors; others, such as miR-21 and the miR-17ϳ92 cluster (16,17), are oncogenic. Some miRNAs (miR-200, miR-31, and miR-10b) regulate metastasis through genes involved in the epithelial-mesenchymal transition (EMT) (18-22). The EMT program is promoted by transcription factors including SNAIL, SLUG, and TWIST and the transcription repressors ZEB1 and ZEB2 (22-24). During EMT, the epithelial marker E-cadherin (CDH1) is downregulated, and the mesenchymal marker vimentin (VIM) is upregulated, resulting in loss of cell-cell contact, increased cell migration, invasion, and metastasis. p21 (p21 Cip1/WAF1 ) is a cyclin-dependent kinase (CDK) inhibitor that suppresses proliferation by inhibiting CDK2 and CDK1 activity at the G 1 /S and G 2 /M transitions (25-28). During the stress response, p21 is transcriptionally upregulated by p53 or by other transcription factors such as E2F1 and MYC (29,30). p21 acts as a CDK inhibitor, but it can also regulate gene expression. Overexpression of p21 decreases the expression levels of cell cycle progression genes and upregulates senescence-inducing genes (31,32). Because p21 is a not a bona fide transcription factor, changes in gene expression mediated by p21 can be explained, in part, by its inhibition of CDKs. However, multiple lines of evidence suggest that p21 acts as a transcription cofactor to directly regulate the activity of important transcription factors, including E2F1 and MYC (25,33).In addition to its role in cell cycle control, p21 has been shown to inhibit EMT in cell lines and in mouse models (34-36). However, the molecular mechanism(s) by which p21 inhibits EMT is not fully understood. In this study, we identified miRNAs regulated by p21 and show that depletion of p21 results in downregulation of several miRNAs, including miR-200 and the miR-183-96-182 (miR-183) cluster, to inhibit EMT, cell migration, and invasion. These findings expand our knowledge about the cell cycle-independent functions of p21 and indicate a key role of a p21-ZEB1 complex in inhibition of EMT through the miR-183 cluster.

The transcription factor caudal-type homeobox 1 (CDX1) is a key regulator of differentiation in the normal colon and in colorectal cancer (CRC). CDX1 activates the expression of enterocyte genes, but it is not clear how the concomitant silencing of stem cell genes is achieved. MicroRNAs (miRNAs) are important mediators of gene repression and have been implicated in tumor suppression and carcinogenesis, but the roles of miRNAs in differentiation, particularly in CRC, remain poorly understood. Here, we identified microRNA-215 (miR-215) as a direct transcriptional target of CDX1 by using highthroughput small RNA sequencing to profile miRNA expression in two pairs of CRC cell lines: CDX1-low HCT116 and HCT116 with stable CDX1 overexpression, and CDX1-high LS174T and LS174T with stable CDX1 knockdown. Validation of candidate miRNAs identified by RNA-seq in a larger cell-line panel revealed miR-215 to be most significantly correlated with CDX1 expression. Quantitative ChIP-PCR and promoter luciferase assays confirmed that CDX1 directly activates miR-215 transcription. miR-215 expression is depleted in FACS-enriched cancer stem cells compared with unsorted samples. Overexpression of miR-215 in poorly differentiated cell lines causes a decrease in clonogenicity, whereas miR-215 knockdown increases clonogenicity and impairs differentiation in CDX1-high cell lines. We identified the genome-wide targets of miR-215 and found that miR-215 mediates the repression of cell cycle and stemness genes downstream of CDX1. In particular, the miR-215 target gene BMI1 has been shown to promote stemness and self-renewal and to vary inversely with CDX1. Our work situates miR-215 as a link between CDX1 expression and BMI1 repression that governs differentiation in CRC.T he caudal-type homeobox 1 (CDX1) transcription factor controls enterocyte differentiation in the colon, where its expression is excluded from the crypt-base stem cell compartment. CDX1 is also central to the capacity of a colorectal cancer (CRC) cell line to differentiate, and it is a negative marker of CRC stem cells (1-3). In 19% of CRC cell lines assayed in a large study, CDX1 expression was completely lost due to promoter methylation and was down-regulated in a further 13% as a result of hemimethylation of the promoter (4). Comparison of CDX1 expression in colorectal adenocarcinoma versus matched normal tissue showed downregulation of CDX1 in 73% of tumors, which was also attributable to promoter methylation (5). Expression of CDX1 also correlates inversely with that of the polycomb complex protein BMI1, which is necessary for the maintenance of quiescent injury-inducible stem cells in the normal crypt and is expressed in cancer stem cells (2, 6-8). The mechanism underlying this inverse correlation has not yet been elucidated.In addition to these correlative data, CDX1 has also been shown to promote directly the expression of structural proteins important for epithelial differentiation including cytokeratin 20 (KRT20) (1), villin (VIL) (9), and FABP1 (10). Introduction...

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Most p53 mutations in human cancers are missense mutations resulting in a full-length mutant p53 protein. Besides losing tumor suppressor activity, some hotspot p53 mutants gain oncogenic functions. This effect is mediated in part, through gene expression changes due to inhibition of p63 and p73 by mutant p53 at their target gene promoters. Here, we report that the tumor suppressor microRNA let-7i is downregulated by mutant p53 in multiple cell lines expressing endogenous mutant p53. In breast cancer patients, significantly decreased let-7i levels were associated with missense mutations in p53. Chromatin immunoprecipitation and promoter luciferase assays established let-7i as a transcriptional target of mutant p53 through p63. Introduction of let-7i to mutant p53 cells significantly inhibited migration, invasion and metastasis by repressing a network of oncogenes including E2F5, LIN28B, MYC and NRAS. Our findings demonstrate that repression of let-7i expression by mutant p53 has a key role in enhancing migration, invasion and metastasis.

BackgroundStalled replication forks at common fragile sites are a major cause of genomic instability. RecQ helicases, a highly conserved family of DNA-unwinding enzymes, are believed to ease ‘roadblocks’ that pose challenge to replication fork progression. Among the five known RecQ homologs in humans, functions of RECQ1, the most abundant of all, are poorly understood. We previously determined that RECQ1 helicase preferentially binds and unwinds substrates that mimic DNA replication/repair intermediates, and interacts with proteins involved in DNA replication restart mechanisms.MethodWe have utilized chromatin immunoprecipitation followed by quantitative real-time PCR to investigate chromatin interactions of RECQ1 at defined genetic loci in the presence or absence of replication stress. We have also tested the sensitivity of RECQ1-depleted cells to aphidicolin induced replication stress.ResultsRECQ1 binds to the origins of replication in unperturbed cells. We now show that conditions of replication stress induce increased accumulation of RECQ1 at the lamin B2 origin in HeLa cells. Consistent with a role in promoting fork recovery or repair, RECQ1 is specifically enriched at two major fragile sites FRA3B and FRA16D where replication forks have stalled following aphidicolin treatment. RECQ1-depletion results in attenuated checkpoint activation in response to replication stress, increased sensitivity to aphidicolin and chromosomal instability.ConclusionsGiven a recent biochemical observation that RECQ1 catalyzes strand exchange on stalled replication fork structures in vitro, our results indicate that RECQ1 facilitates repair of stalled or collapsed replication forks and preserves genome integrity. Our findings provide the first evidence of a crucial role for RECQ1 at naturally occurring fork stalling sites and implicate RECQ1 in mechanisms underlying common fragile site instability in cancer.

The RECQ protein family of helicases has critical roles in protecting and stabilizing the genome. Three of the 5 known members of the human RecQ family are genetically linked with cancer susceptibility syndromes, but the association of the most abundant human RecQ homolog, RECQ1, with cellular transformation is yet unclear. RECQ1 is overexpressed in a variety of human cancers, indicating oncogenic functions. Here, we assessed genome-wide changes in gene expression upon knockdown of RECQ1 in HeLa and MDA-MB-231 cells. Pathway analysis suggested that RECQ1 enhances the expression of multiple genes that play key roles in cell migration, invasion, and metastasis, including EZR, ITGA2, ITGA3, ITGB4, SMAD3, and TGFBR2. Consistent with these results, silencing RECQ1 significantly reduced cell migration and invasion. In comparison to genome-wide annotated promoter regions, the promoters of genes downregulated upon RECQ1 silencing were significantly enriched for a potential G4 DNA forming sequence motif. Chromatin immunoprecipitation assays demonstrated binding of RECQ1 to the G4 motifs in the promoters of select genes downregulated upon RECQ1 silencing. In breast cancer patients, the expression of a subset of RECQ1-activated genes positively correlated with RECQ1 expression. Moreover, high RECQ1 expression was associated with poor prognosis in breast cancer. Collectively, our findings identify a novel function of RECQ1 in gene regulation and indicate that RECQ1 contributes to tumor development and progression, in part, by regulating the expression of key genes that promote cancer cell migration, invasion and metastasis.

Lipoproteins are emulsion particles that consist of lipids and apolipoproteins. Their natural function is to transport lipids and͞or cholesterol to different tissues. We have taken advantage of the hydrophobic interior of these natural emulsions to solubilize DNA. Negatively charged DNA was first complexed with cationic lipids containing a quaternary amine head group. The resulting hydrophobic complex was extracted by chloroform and then incorporated into reconstituted chylomicron remnant particles (Ϸ100 nm in diameter) with an efficiency Ϸ65%. When injected into the portal vein of mice, there were Ϸ5 ng of a transgene product (luciferase) produced per mg of liver protein per 100 g injected DNA. This level of transgene expression was Ϸ100-fold higher than that of mice injected with naked DNA. However, such a high expression was not found after tail vein injection. Histochemical examination revealed that a large number of parenchymal cells and other types of cells in the liver expressed the transgene. Gene expression in the liver increased with increasing injected dose, and was nearly saturated with 50 g DNA. At this dose, the expression was kept at high level in the liver for 2 days and then gradually reduced and almost disappeared by 7 days. However, by additional injection at day 7, gene expression in the liver was completely restored. By injection of plasmid DNA encoding human ␣1-antitrypsin, significant concentrations of hAAT were detected in the serum of injected animals. This is the first nonviral vector that resembles a natural lipoprotein carrier.