Jörg H. Leupold

MicroRNA-21 (miR-21) post-transcriptionally downregulates tumor suppressor Pdcd4 and stimulates invasion, intravasation and metastasis in colorectal cancer

Tumor-suppressor Pdcd4 inhibits transformation and invasion and is downregulated in cancers. So far, it has not been studied as to whether miRNAs, suppressing target expression by binding to the 3′-UTR, regulate Pdcd4 or invasion. The present study was conducted to investigate the regulation of Pdcd4, and invasion/intra-vasation, by miRNAs. A bioinformatics search revealed a conserved target-site for miR-21 within the Pdcd4-3′-UTR at 228–249 nt. In 10 colorectal cell lines, an inverse correlation of miR-21 and Pdcd4-protein was observed. Transfection of Colo206f-cells with miR-21 significantly suppressed a luciferase-reporter containing the Pdcd4-3′-UTR, whereas transfection of RKO with anti-miR-21 increased activity of this construct. This was abolished when a construct mutated at the miR-21/nt228–249 target site was used instead. Anti-miR-21-transfected RKO cells showed an increase of Pdcd4-protein and reduced invasion. Moreover, these cells showed reduced intra-vasation and lung metastasis in a chicken–embryo–metastasis assay. In contrast, overexpression of miR-21 in Colo206f significantly reduced Pdcd4-protein amounts and increased invasion, while Pdcd4-mRNA was unaltered. Resected normal/tumor tissues of 22 colorectal cancer patients demonstrated an inverse correlation between miR-21 and Pdcd4-protein. This is the first study to show that Pdcd4 is negatively regulated by miR-21. Furthermore, it is the first report to demonstrate that miR-21 induces invasion/intravasation/metastasis.

Tumor suppressor Pdcd4 inhibits invasion/intravasation and regulates urokinase receptor (u-PAR) gene expression via Sp-transcription factors

Tumor suppressor Pdcd4 has recently been shown to inhibit invasion by activating activator protein-1 (AP-1); however, little is known of the functionally significant Pdcd4-target genes. The urokinase receptor (u-PAR) promotes invasion/metastasis, and is associated with poor cancer-patient survival. The present study was conducted (1) to investigate a role for Pdcd4 in intravasation, invasion and u-PAR regulation, and (2) to describe mechanisms by which this is achieved. Fourteen cell lines showed reciprocal expression of u-PAR/Pdcd4. Resected tumor/normal tissues of 29 colorectal cancer patients demonstrated a significant inverse correlation between Pdcd4/u-PAR. siRNA-Pdcd4-transfected GEO cells significantly increased endogenous u-PAR mRNA/protein. A u-PAR-promoter-chloramphenicol acetyl transferase (CAT)-reporter was reduced in activity with increasing Pdcd4 expression in RKO. Deletion of a putative Sp-1-binding site (−402/−350) inhibited u-PAR promoter regulation by Pdcd4, this being paralleled by a reduction of Sp1 binding to this region in pdcd4-transfected cells. Pdcd4-transfected cells showed an increase in Sp3 binding to u-PAR promoter region −152/−135, the deletion of which reduces the ability of Pdcd4 to suppress u-PAR promoter activity. Surprisingly, the u-PAR-AP-1 site was not targeted by Pdcd4. Finally, RKO cells overexpressing Pdcd4 showed an inhibition of invasion/intravasation (chicken embryo metastasis assay). These data suggest Pdcd4 as a new negative regulator of intravasation, and qas the invasion-related gene u-PAR. It is the first study to implicate Pdcd4 regulation of gene expression via Sp1/Sp3.

TaqMan ® -based quantification of invasive cells in the chick embryo metastasis assay

The chick embryonic metastasis (CEM) assay is a fast in vivo method to investigate the invasive properties of tumor cells. Until now, most quantification methods were semiquantitative and time-consuming. Here we describe a rapid quantification method using TaqMan technology to quantify the invaded tumor cells in the chorioallantoic membrane of fertilized eggs. This method is based on specific detection of human ALU sequences. Moreover, it provides high sensitivity over a wide linearity range.

MicroRNAs as novel targets and tools in cancer therapy.

MicroRNAs (miRNAs) are currently experiencing a renewed peak of attention not only as diagnostics but also especially as highly promising novel targets or tools for clinical therapy in several different malignant diseases. Moreover, the recent discovery of competing endogenous RNAs (ceRNAs) as novel miRNA-regulators has contributed exciting insights in this regard. Therefore, this review summarizes and discusses the latest findings on (1) how miRNAs have become therapeutic targets of diverse synthetic antagonists, (2) how novel endogenous regulators of miRNAs such as ceRNAs or pseudogenes could emerge as therapeutics scavenging oncogenic miRNAs and (3) how miRNAs themselves are already, and will increasingly be, used as therapeutics. Recent advances on the importance of miRNA-target affinity and the subcellular localization of miRNAs are also discussed. The potential of these developments in different tumor entities and particular hallmarks of cancer such as metastasis, disease progression, interactions with the tumor microenvironment, or cancer stem cells are equally highlighted.

MicroRNA Regulation of Epithelial to Mesenchymal Transition

Epithelial to mesenchymal transition (EMT) is a central regulatory program that is similar in many aspects to several steps of embryonic morphogenesis. In addition to its physiological role in tissue repair and wound healing, EMT contributes to chemo resistance, metastatic dissemination and fibrosis, amongst others. Classically, the morphological change from epithelial to mesenchymal phenotype is characterized by the appearance or loss of a group of proteins which have come to be recognized as markers of the EMT process. As with all proteins, these molecules are controlled at the transcriptional and translational level by transcription factors and microRNAs, respectively. A group of developmental transcription factors form the backbone of the EMT cascade and a large body of evidence shows that microRNAs are heavily involved in the successful coordination of mesenchymal transformation and vice versa, either by suppressing the expression of different groups of transcription factors, or otherwise acting as their functional mediators in orchestrating EMT. This article dissects the contribution of microRNAs to EMT and analyzes the molecular basis for their roles in this cellular process. Here, we emphasize their interaction with core transcription factors like the zinc finger enhancer (E)-box binding homeobox (ZEB), Snail and Twist families as well as some pluripotency transcription factors.

Src induces urokinase receptor gene expression and invasion/intravasation via activator protein-1/p-c-Jun in colorectal cancer.

The urokinase receptor [urokinase plasminogen activator receptor (u-PAR)] promotes invasion and metastasis and is associated with poor patient survival. Recently, it was shown that Src induces u-PAR gene expression via Sp1 bound to the u-PAR promoter region −152/−135. However, u-PAR is regulated by diverse promoter motifs, among them being an essential activator protein-1 (AP-1) motif at −190/−171. Moreover, an in vivo relevance of Src-induced transcriptional regulators of u-PAR–mediated invasion, in particular intravasation, and a relevance in resected patient tumors have not sufficiently been shown. The present study was conducted (a) to investigate if, in particular, AP-1–related transcriptional mediators are required for Src-induced u-PAR–gene expression, (b) to show in vivo relevance of AP-1–mediated Src-induced u-PAR gene expression for invasion/intravasation and for resected tissues from colorectal cancer patients. Src stimulation of the u-PAR promoter deleted for AP-1 region −190/−171 was reduced as compared with the wild-type promoter in cultured colon cancer cells. In gelshifts/chromatin immunoprecipitation, Src-transfected SW480 cells showed an increase of phospho–c-Jun, in addition to JunD and Fra-1, bound to region −190/−171. Src-transfected cells showed a significant increase in c-Jun phosphorylated at Ser73 and also Ser63, which was paralleled by increased phospho–c-jun-NH2-kinase. Significant decreases of invasion/in vivo intravasation (chorionallantoic membrane model) were observed in Src-overexpressing cells treated with Src inhibitors, u-PAR–small interfering RNA, and dominant negative c-Jun (TAM67). In resected tissues of 20 colorectal cancer patients, a significant correlation between Src activity, AP-1 complexes bound to u-PAR region −190/−171, and advanced pN stage were observed. These data suggest that Src-induced u-PAR gene expression and invasion/intravasation in vivo is also mediated via AP-1 region −190/−171, especially bound with c-Jun phosphorylated at Ser73/63, and that this pathway is biologically relevant for colorectal cancer patients, suggesting therapeutic potential. (Mol Cancer Res 2007;5(5):485–96)

NRF-1, and AP-1 regulate the promoter of the human calpain small subunit 1 (CAPNS1) gene

Ubiquitously expressed micro- and m-calpain are cysteine proteases with broad functions in cell spreading, migration, proliferation, apoptosis, and in tumor invasion. They are heterodimers, with a distinct large 80-kDa catalytic, and a common small 28-kDa regulatory subunit (Capn4/CAPNS1). CAPNS1 is required to maintain stability and activity of both calpains. Despite its biological importance, the transcriptional regulation of this gene has not been studied, and the CAPNS1 promoter has not yet been characterized. In this study, we identified the main transcriptional start site, and cloned and characterized the ~2.0 kb upstream region of the CAPNS1 gene. Deletion analysis identified the core promoter located within region -187/+174. Site-directed mutagenesis, EMSA- and supershift analysis identified Sp1-, NRF-1-, and AP-1-binding elements within the CAPNS1 core promoter. Binding of NRF-1, Sp1 and AP-1 to the natural core promoter was confirmed by chromatin immunoprecipitation (ChIP). Site-directed mutagenesis at the NRF-1 site in HeLa and MCF7 cells substantially reduced core promoter activity by 70%, whereas mutation of the AP-1-binding and Sp1-binding site reduced promoter activity by 50% and 30%, respectively. Double mutation of the NRF-1 and the AP-1 site reduced promoter activity by 90%. In Drosophila SL2 cells, ectopic expression of NRF-1 led to a significant induction of CAPNS1 promoter activity. Furthermore, an siRNA against NRF-1 substantially reduced promoter activity in HeLa cells, which was paralleled by a significant downregulation of CAPNS1 mRNA. These results reveal that especially NRF-1, along with AP-1 and, to a minor extent, an Sp1 site, is essential for human CAPNS1 promoter activity and gene expression.

The Disparate Twins: A Comparative Study of CXCR4 and CXCR7 in SDF-1α–Induced Gene Expression, Invasion and Chemosensitivity of Colon Cancer

Purpose: In colorectal cancer, increased expression of the CXC chemokine receptor 4 (CXCR4) has been shown to provoke metastatic disease due to the interaction with its ligand stromal cell-derived factor-1 (SDF-1). Recently, a second SDF-1 receptor, CXCR7, was found to enhance tumor growth in solid tumors. Albeit signaling cascades via SDF-1/CXCR4 have been intensively studied, the significance of the SDF-1/CXCR7–induced intracellular communication triggering malignancy is still only marginally understood. Experimental Design: In tumor tissue of 52 patients with colorectal cancer, we observed that expression of CXCR7 and CXCR4 increased with tumor stage and tumor size. Asking whether activation of CXCR4 or CXCR7 might result in a similar expression pattern, we performed microarray expression analyses using lentivirally CXCR4- and/or CXCR7-overexpressing SW480 colon cancer cell lines with and without stimulation by SDF-1α. Results: Gene regulation via SDF-1α/CXCR4 and SDF-1α/CXCR7 was completely different and partly antidromic. Differentially regulated genes were assigned by gene ontology to migration, proliferation, and lipid metabolic processes. Expressions of AKR1C3, AXL, C5, IGFBP7, IL24, RRAS, and TNNC1 were confirmed by quantitative real-time PCR. Using the in silico gene set enrichment analysis, we showed that expressions of miR-217 and miR-218 were increased in CXCR4 and reduced in CXCR7 cells after stimulation with SDF-1α. Functionally, exposure to SDF-1α increased invasiveness of CXCR4 and CXCR7 cells, AXL knockdown hampered invasion. Compared with controls, CXCR4 cells showed increased sensitivity against 5-FU, whereas CXCR7 cells were more chemoresistant. Conclusions: These opposing results for CXCR4- or CXCR7-overexpressing colon carcinoma cells demand an unexpected attention in the clinical application of chemokine receptor antagonists such as plerixafor. Clin Cancer Res; 20(3); 604–16. ©2013 AACR.

PMA up-regulates the transcription of Axl by AP-1 transcription factor binding to TRE sequences via the MAPK cascade in leukaemia cells

Background. Axl is a receptor tyrosine kinase promoting anti‐apoptosis, invasion and mitogenesis, and is highly expressed in different solid cancers. Axl basal transcriptional activity is driven by Sp1/Sp3, and overexpression of MZF‐1 (myeloid zinc‐finger 1) induces Axl transcription and gene expression. Furthermore, Axl expression is epigenetically controlled by CpG hypermethylation; however, little is known about inducible Axl gene expression and Axl regulation in haematopoetic malignancies.

A Systematic Approach to Defining the microRNA Landscape in Metastasis

The microRNA (miRNA) landscape changes during the progression of cancer. We defined a metastasis-associated miRNA landscape using a systematic approach. We profiled and validated miRNA and mRNA expression in a unique series of human colorectal metastasis tissues together with their matched primary tumors and corresponding normal tissues. We identified an exclusive miRNA signature that is differentially expressed in metastases. Three of these miRNAs were identified as key drivers of an EMT-regulating network acting though a number of novel targets. These targets include SIAH1, SETD2, ZEB2, and especially FOXN3, which we demonstrated for the first time as a direct transcriptional suppressor of N-cadherin. The modulation of N-cadherin expression had significant impact on migration, invasion, and metastasis in two different in vivo models. The significant deregulation of the miRNAs defining the network was confirmed in an independent patient set as well as in a database of diverse malignancies derived from more than 6,000 patients. Our data define a novel metastasis-orchestrating network based on systematic hypothesis generation from metastasis tissues.