Although constitutive activation of beta-catenin/Tcf signalling is implicated in the development of human cancers, the mechanisms by which the beta-catenin/Tcf pathway promotes tumorigenesis are incompletely understood. Messenger RNA turnover has a major function in regulating gene expression and is responsive to developmental and environmental signals. mRNA decay rates are dictated by cis-acting elements within the mRNA and by trans-acting factors, such as RNA-binding proteins (reviewed in refs 2, 3). Here we show that beta-catenin stabilizes the mRNA encoding the F-box protein betaTrCP1, and identify the RNA-binding protein CRD-BP (coding region determinant-binding protein) as a previously unknown target of beta-catenin/Tcf transcription factor. CRD-BP binds to the coding region of betaTrCP1 mRNA. Overexpression of CRD-BP stabilizes betaTrCP1 mRNA and elevates betaTrCP1 levels (both in cells and in vivo), resulting in the activation of the Skp1-Cullin1-F-box protein (SCF)(betaTrCP) E3 ubiquitin ligase and in accelerated turnover of its substrates including IkappaB and beta-catenin. CRD-BP is essential for the induction of both betaTrCP1 and c-Myc by beta-catenin signalling in colorectal cancer cells. High levels of CRD-BP that are found in primary human colorectal tumours exhibiting active beta-catenin/Tcf signalling implicates CRD-BP induction in the upregulation of betaTrCP1, in the activation of dimeric transcription factor NF-kappaB and in the suppression of apoptosis in these cancers.

miRNAs are largely known to base-pair with the 3′UTR of target mRNAs, downregulating their stability and translation. mRNA of βTrCP1 ubiquitin ligase is very unstable, but unlike the majority of mRNAs where 3′UTR determines the rate of mRNA turnover, βTrCP1 mRNA contains cis-acting destabilizing elements within its coding region. Here we show that degradation of mRNA of βTrCP1 is miRNA-dependent, and identified miR-183 as a microRNA that interacts with the coding region of βTrCP1 mRNA. Argonaute2 interacts with the same region of βTrCP1 mRNA in miR-183-dependent manner. Inhibition of mir-183 function or disruption of mir-183-binding site stabilizes βTrCP1 mRNA and elevates βTrCP1 levels, resulting in activation of the SCFβTrCP E3 ubiquitin ligase. We have previously shown that RNA-binding protein, CRD-BP, binds to the coding region of βTrCP1 mRNA and stabilizes it. In this report we demonstrate that CRD-BP prevents degradation of βTrCP1 mRNA by attenuating its miR-183-dependent interaction with Ago2.

Advancing pluripotent stem cell technologies for modeling hematopoietic stem cell development and blood therapies requires identifying key regulators of hematopoietic commitment from human pluripotent stem cells (hPSCs). Here, by screening the effect of 27 candidate factors, we reveal two groups of transcriptional regulators capable of inducing distinct hematopoietic programs from hPSCs: panmyeloid (ETV2 and GATA2) and erythro-megakaryocytic (GATA2 and TAL1). In both cases, these transcription factors directly convert hPSCs to endothelium, which subsequently transforms into blood cells with pan-myeloid or erythromegakaryocytic potential. These data demonstrate that two distinct genetic programs regulate the hematopoietic development from hPSCs and that both of these programs specify hPSCs directly to hemogenic endothelial cells. Additionally, this study provides a novel method for the efficient induction of blood and endothelial cells from hPSCs via overexpression of modified mRNA for the selected transcription factors.

We previously showed that the calcium-binding protein S100A4 is overexpressed during the progression of prostate cancer (CaP) in humans and in the TRAMP (transgenic adenocarcinoma of the mouse prostate) mouse model. We tested a hypothesis that the S100A4 gene plays a role in the invasiveness of human CaP and may be associated with its metastatic spread. We observed that siRNAmediated suppression of the S100A4 gene significantly reduced the proliferative and invasive capability of the highly invasive CaP cells PC-3. We evaluated the mechanism through which the S100A4 gene controls invasiveness of cells by using a macroarray containing 96 well characterized metastatic genes. We found that matrix metalloproteinase 9 (MMP-9) and its tissue inhibitor (TIMP-1) were highly responsive to S100A4 gene suppression. Furthermore, S100A4 suppression significantly reduced the expression and proteolytic activity of MMP-9. By employing an MMP-9-promoter reporter, we observed a significant reduction in the transcriptional activation of the MMP-9 gene in S100A4-siRNA-transfected cells. Cells overexpressing the S100A4 gene (when transfected with pcDNA3.1-S100A4 plasmid) also significantly expressed MMP-9 and TIMP-1 genes with increased proteolytic activity of MMP-9 concomitant to increased transcriptional activation of the MMP-9 gene. S100A4-siRNA-transfected cells exhibited a reduced rate of tumor growth under in vivo conditions. Our data demonstrate that the S100A4 gene controls the invasive potential of human CaP cells through regulation of MMP-9 and that this association may contribute to metastasis of CaP cells. We suggest that S100A4 could be used as a biomarker for CaP progression and a novel therapeutic or chemopreventive target for human CaP treatment. extracellular matrix ͉ metastasis ͉ biomarker A pproximately 27,350 prostate cancer (CaP)-related deaths are predicted during this year alone in the United States, and despite recent improvements in diagnostic and therapeutic techniques, the survival rate of CaP patients is poor because of the posttreatment recurrence of disease (1, 2). The lack of effective therapies for advanced CaP is related to a large extent to poor understanding of the molecular mechanisms underlying the progression of disease toward invasion and metastasis (3). Thus, the identification of new predictive biomarkers, especially those that are indicative of invasiveness of the disease, which could serve as targets for establishing effectiveness of therapeutic and chemopreventive interventions, will improve clinical management of CaP. S100A4 (also known as mts1), a calcium-binding protein associated with invasion and metastasis of cancer cells, has been reported to be frequently overexpressed in metastatic tumors, normal cells with uninhibited movement (such as macrophages), and transformed cells, and in various cancer types such as breast, ovary, thyroid, lung, esophageal squamous cell carcinoma, gastric, and colon (4-6). Studies have shown that breast cancers expressing high levels of S100A4 have a sign...

The Hedgehog (Hh) signaling pathway plays a crucial role in embryogenesis and has been linked to the development of several human malignancies. The transcription factor Gli2 plays a key role in the transduction of Hh signals by modulating transcription of some Hh target genes, yet the mechanisms that control Gli2 protein expression are largely unknown. Here we report that ␤-transducin repeat-containing protein (␤-TrCP) E3 ubiquitin ligase is required for Gli2 degradation. ␤-TrCP2 directly binds wild type Gli2 and promotes its ubiquitination. Single amino acid substitution in Gli2 putative binding site inhibits its interaction with ␤-TrCP2, its ubiquitination, and stabilizes the Gli2 protein. Stable Gli2 mutant is expressed in higher levels and is more potent in the activation of Gli-dependent transcription as compared with wild type Gli2. We also found that GLI2 protein is expressed highly in prostate cancer cell lines and primary tumors, whereas the level of GLI2 mRNA is not appreciably different in normal and neoplastic prostate. These data identify ␤-TrCP2 as a pivotal regulator of Gli2 expression and point to an important role for posttranslational modulation of GLI2 protein levels in Hh pathway-associated human prostate cancer.