Irina Elcheva

S100A4 accelerates tumorigenesis and invasion of human prostate cancer through the transcriptional regulation of matrix metalloproteinase 9

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 siRNA-mediated 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.

CRD-BP mediates stabilization of βTrCP1 and c- myc mRNA in response to β-catenin signalling

Although constitutive activation of β-catenin/Tcf signalling is implicated in the development of human cancers, the mechanisms by which the β-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 β-catenin stabilizes the mRNA encoding the F-box protein βTrCP1, and identify the RNA-binding protein CRD-BP (coding region determinant-binding protein) as a previously unknown target of β-catenin/Tcf transcription factor. CRD-BP binds to the coding region of βTrCP1 mRNA. Overexpression of CRD-BP stabilizes βTrCP1 mRNA and elevates βTrCP1 levels (both in cells and in vivo), resulting in the activation of the Skp1-Cullin1-F-box protein (SCF)βTrCP E3 ubiquitin ligase and in accelerated turnover of its substrates including IκB and β-catenin. CRD-BP is essential for the induction of both βTrCP1 and c-Myc by β-catenin signalling in colorectal cancer cells. High levels of CRD-BP that are found in primary human colorectal tumours exhibiting active β-catenin/Tcf signalling implicates CRD-BP induction in the upregulation of βTrCP1, in the activation of dimeric transcription factor NF-κB and in the suppression of apoptosis in these cancers.

Gli2 Is Targeted for Ubiquitination and Degradation by β-TrCP Ubiquitin Ligase

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 β-transducin repeat-containing protein (β-TrCP) E3 ubiquitin ligase is required for Gli2 degradation. β-TrCP2 directly binds wild type Gli2 and promotes its ubiquitination. Single amino acid substitution in Gli2 putative binding site inhibits its interaction with β-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 β-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.

Direct induction of haematoendothelial programs in human pluripotent stem cells by transcriptional regulators

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.

Overexpression of mRNA-binding protein CRD-BP in malignant melanomas

Wnt/β-catenin signaling pathway plays an important role in embryogenesis, stem cell maintenance, tumorigenesis and aging. Here, we show that RNA-binding protein, coding region determinant-binding protein (CRD-BP) (a transcriptional target of Wnt signaling pathway), is highly expressed in primary human malignant melanomas and melanoma cell lines with activated Wnt/β-catenin signaling pathway. Upregulation of CRD-BP is associated with an elevated level of β-TrCP1 ubiquitin ligase receptor and activation of nuclear transcriptional factors-kappa B (NF-κB) signaling. Knockdown of CRD-BP inhibits NF-κB activity, induces apoptosis, and suppresses proliferation and tumorigenic properties of melanoma cells.

Somatic genetic alterations (loh) in benign, borderline and invasive ovarian tumours: Intratumoral molecular heterogeneity

Loss of heterozygosity (LOH) affects a number of chromosome regions in ovarian cancer, pointing to the possible involvement of tumour‐suppressor genes in ovarian tumorigenesis. We performed comparative analysis of allelic loss at 6 frequently affected chromosome regions in a panel of 53 benign, borderline and malignant ovarian tumours. Precursor lesions could provide evidence that an accumulation of genetic events is required for normal ovarian epithelium to generate malignant tumours. LOH on chromosome 1p was relatively common in benign, borderline and malignant tumours, while at 11p and 7q it was observed not only in invasive but also in borderline tumours. Moreover, 17q and 18q were affected mainly in advanced malignant tumours and revealed a high frequency of clonal intratumoral heterogeneity. We encountered different spectra of genetic alterations in primary tumours and their metastasis, which may be the results of intratumoral heterogeneity leading to dissemination in only some sub‐clones. Int. J. Cancer 82:822–826, 1999.

Cytokine-free directed differentiation of human pluripotent stem cells efficiently produces hemogenic endothelium with lymphoid potential

BackgroundThe robust generation of human hematopoietic progenitor cells from induced or embryonic pluripotent stem cells would be beneficial for multiple areas of research, including mechanistic studies of hematopoiesis, the development of cellular therapies for autoimmune diseases, induced transplant tolerance, anticancer immunotherapies, disease modeling, and drug/toxicity screening. Over the past years, significant progress has been made in identifying effective protocols for hematopoietic differentiation from pluripotent stem cells and understanding stages of mesodermal, endothelial, and hematopoietic specification. Thus, it has been shown that variations in cytokine and inhibitory molecule treatments in the first few days of hematopoietic differentiation define primitive versus definitive potential of produced hematopoietic progenitor cells. The majority of current feeder-free, defined systems for hematopoietic induction from pluripotent stem cells include prolonged incubations with various cytokines that make the differentiation process complex and time consuming. We established that the application of Wnt agonist CHIR99021 efficiently promotes differentiation of human pluripotent stem cells in the absence of any hematopoietic cytokines to the stage of hemogenic endothelium capable of definitive hematopoiesis.MethodsThe hemogenic endothelium differentiation was accomplished in an adherent, serum-free culture system by applying CHIR99021. Hemogenic endothelium progenitor cells were isolated on day 5 of differentiation and evaluated for their endothelial, myeloid, and lymphoid potential.ResultsMonolayer induction based on GSK3 inhibition, described here, yielded a large number of CD31+CD34+ hemogenic endothelium cells. When isolated and propagated in adherent conditions, these progenitors gave rise to mature endothelium. When further cocultured with OP9 mouse stromal cells, these progenitors gave rise to various cells of myeloid lineages as well as natural killer lymphoid, T-lymphoid, and B-lymphoid cells.ConclusionThe results of this study substantiate a method that significantly reduces the complexity of current protocols for hematopoietic induction, offers a defined system to study the factors that affect the early stages of hematopoiesis, and provides a new route of lymphoid and myeloid cell derivation from human pluripotent stem cells, thus enhancing their use in translational medicine.

Direct Induction of Hemogenic Endothelium and Blood by Overexpression of Transcription Factors in Human Pluripotent Stem Cells.

During development, hematopoietic cells arise from a specialized subset of endothelial cells, hemogenic endothelium (HE). Modeling HE development in vitro is essential for mechanistic studies of the endothelial-hematopoietic transition and hematopoietic specification. Here, we describe a method for the efficient induction of HE from human pluripotent stem cells (hPSCs) by way of overexpression of different sets of transcription factors. The combination of ETV2 and GATA1 or GATA2 TFs is used to induce HE with pan-myeloid potential, while a combination of GATA2 and TAL1 transcription factors allows for the production of HE with erythroid and megakaryocytic potential. The addition of LMO2 to GATA2 and TAL1 combination substantially accelerates differentiation and increases erythroid and megakaryocytic cells production. This method provides an efficient and rapid means of HE induction from hPSCs and allows for the observation of the endothelial-hematopoietic transition in a culture dish. The protocol includes hPSCs transduction procedures and post-transduction analysis of HE and blood progenitors.

Application of small molecule CHIR99021 leads to the loss of hemangioblast progenitor and increased hematopoiesis of human pluripotent stem cells

Improving our understanding of the intricacies of hematopoietic specification of induced or embryonic human pluripotent stem cells is beneficial for many areas of research and translational medicine. Currently, it is not clear whether, during human pluripotent stem cells hematopoietic differentiation in vitro, the maturation of definitive progenitors proceeds through a primitive progenitor (hemangioblast) intermediate or if it develops independently. The objective of this study was to investigate the early stages of hematopoietic specification of pluripotent stem cells in vitro. By implementing an adherent culture, serum-free differentiation system that utilizes a small molecule, CHIR99021, to induce human pluripotent stem cells toward various hematopoietic lineages, we established that, compared with the OP9 coculture hematopoietic induction system, the application of CHIR99021 alters the early steps of hematopoiesis such as hemangioblasts, angiogenic hematopoietic progenitors, and hemogenic endothelium. Importantly, it is associated with the loss of hemangioblast progenitors, loss of CD43+ (primitive hematopoietic marker) expression, and predominant development of blast-forming unit erythroid colonies in semisolid medium. These data support the hypothesis that the divergence of primitive and definitive programs during human pluripotent stem cells differentiation precedes the hemangioblast stage. Furthermore, we have shown that the inhibition of primitive hematopoiesis is associated with an increase in hematopoietic potential, which is a fruitful finding due to the growing need for lymphoid and myeloid cells in translational applications.