The Interaction between the Forkhead Thyroid Transcription Factor TTF-2 and the Constitutive Factor CTF/NF-1 Is Required for Efficient Hormonal Regulation of the Thyroperoxidase Gene Transcription

The forkhead thyroid-specific transcription factor TTF-2 is the main mediator of thyrotropin and insulin regulation of thyroperoxidase (TPO) gene expression. This function depends on multimerization and specific orientation of its DNA-binding site, suggesting that TTF-2 is part of a complex interaction network within the TPO promoter. This was confirmed by transfection experiments and by protein-DNA interaction studies, which demonstrated that CTF/NF1 proteins bind 10 base pairs upstream of the TTF-2- binding site to enhance its action in hormone-induced expression of the TPO gene. GST pull-down assays showed that TTF-2 physically interacts with CTF/NF1 proteins. In addition, we demonstrate that increasing the distance between both transcription factors binding sites by base pair insertion results in loss of promoter activity and in a drastic decrease on the ability of the promoter to respond to the hormones. CTF/NF1 is a family of transcription factors that contributes to constitutive and cell-type specific gene expression. Originally identified as factors implicated in the replication of adenovirus, this group of proteins (CTF/NF1-A, -B, -C, and - X) is now known to be involved in the regulation of several genes. In contrast to other reports regarding the involvement of these proteins in inducible gene expression, we show here that members of this family of transcription factors are regulated by hormones. With the use of specific CTF/NF1 DNA probes and antibodies we demonstrate that CTF/NF1-C is a thyrotropin-, cAMP-, and insulin-inducible protein. Thus CTF/NF1 proteins do not only mediate hormone-induced gene expression cooperating with TTF-2, but are themselves hormonally regulated. All these findings are clearly of important value in understanding the mechanisms governing the transcription regulation of RNA polymerase II promoters, which often contain binding sites for multiple transcription factors

Hedgehog signal activation in oesophageal cancer patients undergoing neoadjuvant chemoradiotherapy

The zinc finger protein glioma-associated oncogene homologue 1 (Gli-1) is a critical component of the Hedgehog (Hh) signalling pathway, which is essential for morphogenesis and stem-cell renewal, and is dysregulated in many cancer types. As data were not available on the role of Gli-1 expression in oesophageal cancer progression, we analysed whether it could be used to predict disease progression and prognosis in oesophageal cancer patients undergoing neoadjuvant chemoradiotherapy (CRT). Among 69 patients with histologically confirmed oesophageal squamous cell carcinomas (ESCCs), 25 showed a pathological complete response after preoperative CRT. Overall survival (OS) was significantly associated with lymph-node metastasis, distant metastasis, and CRT, and was further correlated with the absence of both Gli-1 nuclear expression and residual tumour. All patients with Gli-1 nuclear expression (10.1%) had distant or lymph-node metastasis, and six out of seven died within 13 months. Furthermore, patients with Gli-1 nuclear-positive cancers showed significantly poorer prognoses than those without (disease-free survival: mean DFS time 250 vs 1738 months, 2-year DFS 0 vs 54.9%, P=0.009; OS: mean OS time 386 vs 1742 months, 2-year OS 16.7 vs 54.9%, P=0.001). Our study provides the first evidence that Gli-1 nuclear expression is a strong and independent predictor of early relapse and poor prognosis in ESCC after CRT. These findings suggest that Hh signal activation might promote cancer regrowth and progression after CRT

Integrated Functional, Gene Expression and Genomic Analysis for the Identification of Cancer Targets

The majority of new drug approvals for cancer are based on existing therapeutic targets. One approach to the identification of novel targets is to perform high-throughput RNA interference (RNAi) cellular viability screens. We describe a novel approach combining RNAi screening in multiple cell lines with gene expression and genomic profiling to identify novel cancer targets. We performed parallel RNAi screens in multiple cancer cell lines to identify genes that are essential for viability in some cell lines but not others, suggesting that these genes constitute key drivers of cellular survival in specific cancer cells. This approach was verified by the identification of PIK3CA, silencing of which was selectively lethal to the MCF7 cell line, which harbours an activating oncogenic PIK3CA mutation. We combined our functional RNAi approach with gene expression and genomic analysis, allowing the identification of several novel kinases, including WEE1, that are essential for viability only in cell lines that have an elevated level of expression of this kinase. Furthermore, we identified a subset of breast tumours that highly express WEE1 suggesting that WEE1 could be a novel therapeutic target in breast cancer. In conclusion, this strategy represents a novel and effective strategy for the identification of functionally important therapeutic targets in cancer

Coordinated Translocation of Mammalian Gli Proteins and Suppressor of Fused to the Primary Cilium

Intracellular transduction of Hedgehog (Hh) signals in mammals requires functional primary cilia. The Hh signaling effectors, the Gli family of transcription factors, and their negative regulator, Suppressor of Fused (Sufu), accumulate at the tips of cilia; however, the molecular mechanism regulating this localization remains elusive. In the current study, we show that the ciliary localization of mammalian Gli proteins depends on both their N-terminal domains and a central region lying C-terminal to the zinc-finger DNA-binding domains. Invertebrate Gli homologs Ci and Tra1, when over-expressed in ciliated mouse fibroblasts, fail to localize to the cilia, suggesting the lack of a vertebrate-specific structural feature required for ciliary localization. We further show that activation of protein kinase A (PKA) efficiently inhibits ciliary localization of Gli2 and Gli3, but only moderately affects the ciliary localization of Gli1. Interestingly, variants of Gli2 mimicking the phosphorylated or non-phosphorylated states of Gli2 are both localized to the cilia, and their ciliary localizations are subjected to the inhibitory effect of PKA activation, suggesting a likely indirect mechanism underlying the roles of PKA in Gli ciliary localization. Finally, we show that ciliary localization of Sufu is dependent on ciliary-localized Gli proteins, and is inhibited by PKA activation, suggesting a coordinated mechanism for the ciliary translocation of Sufu and Gli proteins

Discovering cancer genes by integrating network and functional properties

<p>Abstract</p> <p>Background</p> <p>Identification of novel cancer-causing genes is one of the main goals in cancer research. The rapid accumulation of genome-wide protein-protein interaction (PPI) data in humans has provided a new basis for studying the topological features of cancer genes in cellular networks. It is important to integrate multiple genomic data sources, including PPI networks, protein domains and Gene Ontology (GO) annotations, to facilitate the identification of cancer genes.</p> <p>Methods</p> <p>Topological features of the PPI network, as well as protein domain compositions, enrichment of gene ontology categories, sequence and evolutionary conservation features were extracted and compared between cancer genes and other genes. The predictive power of various classifiers for identification of cancer genes was evaluated by cross validation. Experimental validation of a subset of the prediction results was conducted using siRNA knockdown and viability assays in human colon cancer cell line DLD-1.</p> <p>Results</p> <p>Cross validation demonstrated advantageous performance of classifiers based on support vector machines (SVMs) with the inclusion of the topological features from the PPI network, protein domain compositions and GO annotations. We then applied the trained SVM classifier to human genes to prioritize putative cancer genes. siRNA knock-down of several SVM predicted cancer genes displayed greatly reduced cell viability in human colon cancer cell line DLD-1.</p> <p>Conclusion</p> <p>Topological features of PPI networks, protein domain compositions and GO annotations are good predictors of cancer genes. The SVM classifier integrates multiple features and as such is useful for prioritizing candidate cancer genes for experimental validations.</p

Identification of RegIV as a Novel GLI1 Target Gene in Human Pancreatic Cancer

GLI1 is the key transcriptional factor in the Hedgehog signaling pathway in pancreatic cancer. RegIV is associated with regeneration, and cell growth, survival, adhesion and resistance to apoptosis. We aimed to study RegIV expression in pancreatic cancer and its relationship to GLI1.GLI1 and RegIV expression were evaluated in tumor tissue and adjacent normal tissues of pancreatic cancer patients and 5 pancreatic cancer cell lines by qRT-PCR, Western blot, and immunohistochemistry (IHC), and the correlation between them. The GLI1-shRNA lentiviral vector was constructed and transfected into PANC-1, and lentiviral vector containing the GLI1 expression sequence was constructed and transfected into BxPC-3. GLI1 and RegIV expression were evaluated by qRT-PCR and Western blot. Finally we demonstrated RegIV to be the target of GLI1 by chromatin immunoprecipitation (CHIP) and electrophoretic mobility shift assays (EMSA).The results of IHC and qRT-PCR showed that RegIV and GLI1 expression was higher in pancreatic cancer tissues versus adjacent normal tissues (p<0.001). RegIV expression correlated with GLI1 expression in these tissues (R = 0.795, p<0.0001). These results were verified for protein (R = 0.939, p = 0.018) and mRNA expression (R = 0.959, p = 0.011) in 5 pancreatic cancer cell lines. RegIV mRNA and protein expression was decreased (94.7±0.3%, 84.1±0.5%; respectively) when GLI1 was knocked down (82.1±3.2%, 76.7±2.2%; respectively) by the RNAi technique. GLI1 overexpression in mRNA and protein level (924.5±5.3%, 362.1±3.5%; respectively) induced RegIV overexpression (729.1±4.3%, 339.0±3.7%; respectively). Moreover, CHIP and EMSA assays showed GLI1 protein bound to RegIV promotor regions (GATCATCCA) in pancreatic cancer cells.GLI1 promotes RegIV transcription by binding to the RegIV gene promoter in pancreatic cancer

Tumor Associated Macrophages Protect Colon Cancer Cells from TRAIL-Induced Apoptosis through IL-1β- Dependent Stabilization of Snail in Tumor Cells

We recently reported that colon tumor cells stimulate macrophages to release IL-1beta, which in turn inactivates GSK3beta and enhances Wnt signaling in colon cancer cells, generating a self-amplifying loop that promotes the growth of tumor cells.Here we describe that macrophages protect HCT116 and Hke-3 colon cancer cells from TRAIL-induced apoptosis. Inactivation of IL-1beta by neutralizing IL-1beta antibody, or silencing of IL-1beta in macrophages inhibited their ability to counter TRAIL-induced apoptosis. Accordingly, IL-1beta was sufficient to inhibit TRAIL-induced apoptosis. TRAIL-induced collapse of the mitochondrial membrane potential (Delta psi) and activation of caspases were prevented by macrophages or by recombinant IL-1beta. Pharmacological inhibition of IL-1beta release from macrophages by vitamin D(3), a potent chemopreventive agent for colorectal cancer, restored the ability of TRAIL to induce apoptosis of tumor cells cultured with macrophages. Macrophages and IL-1beta failed to inhibit TRAIL-induced apoptosis in HCT116 cells expressing dnIkappaB, dnAKT or dnTCF4, confirming that they oppose TRAIL-induced cell death through induction of Wnt signaling in tumor cells. We showed that macrophages and IL-1beta stabilized Snail in tumor cells in an NF-kappaB/Wnt dependent manner and that Snail deficient tumor cells were not protected from TRAIL-induced apoptosis by macrophages or by IL-1beta, demonstrating a crucial role of Snail in the resistance of tumor cells to TRAIL.We have identified a positive feedback loop between tumor cells and macrophages that propagates the growth and promotes the survival of colon cancer cells: tumor cells stimulate macrophages to secrete IL-1beta, which in turn, promotes Wnt signaling and stabilizes Snail in tumor cells, conferring resistance to TRAIL. Vitamin D(3) halts this amplifying loop by interfering with the release of IL-1beta from macrophages. Accordingly, vitamin D(3) sensitizes tumor cells to TRAIL-induced apoptosis, suggesting that the therapeutic efficacy of TRAIL could be augmented by this readily available chemopreventive agent

CSN-mediated deneddylation differentially modulates Ci155 proteolysis to promote Hedgehog signalling responses

The Hedgehog (Hh) morphogen directs distinct cell responses according to its distinct signalling levels. Hh signalling stabilizes transcription factor cubitus interruptus (Ci) by prohibiting SCFSlimb-dependent ubiquitylation and proteolysis of Ci. How graded Hh signalling confers differential SCFSlimb-mediated Ci proteolysis in responding cells remains unclear. Here, we show that in COP9 signalosome (CSN) mutants, in which deneddylation of SCFSlimb is inactivated, Ci is destabilized in low-to-intermediate Hh signalling cells. As a consequence, expression of the low-threshold Hh target gene dpp is disrupted, highlighting the critical role of CSN deneddylation on low-to-intermediate Hh signalling response. The status of Ci phosphorylation and the level of E1 ubiquitin-activating enzyme are tightly coupled to this CSN regulation. We propose that the affinity of substrate–E3 interaction, ligase activity and E1 activity are three major determinants for substrate ubiquitylation and thereby substrate degradation in vivo

CD155/PVR plays a key role in cell motility during tumor cell invasion and migration

BACKGROUND: Invasion is an important early step of cancer metastasis that is not well understood. Developing therapeutics to limit metastasis requires the identification and validation of candidate proteins necessary for invasion and migration. METHODS: We developed a functional proteomic screen to identify mediators of tumor cell invasion. This screen couples Fluorophore Assisted Light Inactivation (FALI) to a scFv antibody library to systematically inactivate surface proteins expressed by human fibrosarcoma cells followed by a high-throughput assessment of transwell invasion. RESULTS: Using this screen, we have identified CD155 (the poliovirus receptor) as a mediator of tumor cell invasion through its role in migration. Knockdown of CD155 by FALI or by RNAi resulted in a significant decrease in transwell migration of HT1080 fibrosarcoma cells towards a serum chemoattractant. CD155 was found to be highly expressed in multiple cancer cell lines and primary tumors including glioblastoma (GBM). Knockdown of CD155 also decreased migration of U87MG GBM cells. CD155 is recruited to the leading edge of migrating cells where it colocalizes with actin and αv-integrin, known mediators of motility and adhesion. Knockdown of CD155 also altered cellular morphology, resulting in cells that were larger and more elongated than controls when plated on a Matrigel substrate. CONCLUSION: These results implicate a role for CD155 in mediating tumor cell invasion and migration and suggest that CD155 may contribute to tumorigenesis

Travelling and splitting of a wave of hedgehog expression involved in spider-head segmentation

During development segmentation is a process that generates a spatial periodic pattern. Peak splitting of waves of gene expression is a mathematically predicted, simple strategy accounting for this type of process, but it has not been well characterized biologically. Here we show temporally repeated splitting of gene expression into stripes that is associated with head axis growth in the spider Achaearanea embryo. Preceding segmentation, a wave of hedgehog homologue gene expression is observed to travel posteriorly during development stage 6. This stripe, co-expressing an orthodenticle homologue, undergoes two cycles of splitting and shifting accompanied by convergent extension, serving as a generative zone for the head segments. The two orthodenticle and odd-paired homologues are identified as targets of Hedgehog signalling, and evidence suggests that their activities mediate feedback to maintain the head generative zone and to promote stripe splitting in this zone. We propose that the 'stripe-splitting' strategy employs genetic components shared with Drosophila blastoderm subdivision, which are required for participation in an autoregulatory signalling network