Regulation of components of AP-1 transcription factor by early and late Ras signals

Experiments utilising either tumour cells or over-expression of oncogenes like Ras and its potential downstream mediators have yielded a wealth of information over the last decade. Qualitative and quantitative analysis of AP-1 transcription factor has been extensively analysed in response to various oncogenic signals. One basic criticism is that the continued presence of an activated component of cellular signaling renders the study of early Ras-mediated signaling impossible. Inducible systems for oncogene expression offer a valuable alternative for detailed analysis of signal transduction pathways. Here, we report the comparative analysis of components of oncogenic pathways between tumour cells and cells that carry inducible oncogenes

Selective BRAFV600E Inhibitor PLX4720, Requires TRAIL Assistance to Overcome Oncogenic PIK3CA Resistance

Documented sensitivity of melanoma cells to PLX4720, a selective BRAFV600E inhibitor, is based on the presence of mutant BRAFV600E alone, while wt-BRAF or mutated KRAS result in cell proliferation. In colon cancer appearance of oncogenic alterations is complex , since BRAF, like KRAS mutations, tend to co-exist with those in PIK3CA and mutated PI3K has been shown to interfere with the successful application of MEK inhibitors. When PLX4720 was used to treat colon tumours, results were not encouraging and herein we attempt to understand the cause of this recorded resistance and discover rational therapeutic combinations to resensitize oncogene driven tumours to apoptosis. Treatment of two genetically different BRAFV600E mutant colon cancer cell lines with PLX4720 conferred complete resistance to cell death. Even though p-MAPK/ ERK kinase (MEK) suppression was achieved, TRAIL, an apoptosis inducing agent, was used synergistically in order to achieve cell death by apoptosis in RKOBRAFV600E/PIK3CAH1047 cells. In contrast, for the same level of apoptosis in HT29BRAFV600E/PIK3CAP449T cells, TRAIL was combined with 17-AAG, an Hsp90 inhibitor. For cells where PLX4720 was completely ineffective, 17-AAG was alternatively used to target mutant BRAFV600E. TRAIL dependence on the constitutive activation of BRAFV600E is emphasised through the overexpression of BRAFV600E in the permissive genetic background of colon adenocarcinoma Caco-2 cells. Pharmacological suppression of the PI3K pathway further enhances the synergistic effect between TRAIL and PLX4720 in RKO cells, indicating the presence of PIK3CAMT as the inhibitory factor. Another rational combination includes 17-AAG synergism with TRAIL in a BRAFV600E mutant dependent manner to commit cells to apoptosis, through DR5 and the amplification of the apoptotic pathway. We have successfully utilised combinations of two chemically unrelated BRAFV600E inhibitors in combination with TRAIL in a BRAFV600E mutated background and provided insight for new anti-cancer strategies where the activated PI3KCA mutation oncogene should be suppressed

Sensitization of (colon) cancer cells to death receptor related therapies. A report from the FP6-ONCODEATH research consortium

The objective of the ONCODEATH consortium [EU Research Consortium >ONCODEATH> (2006-2010)] was to achieve sensitization of solid tumor cells to death receptor related therapies using rational mechanism-based drug combinations of targeted therapies. In this collaborative effort, during a period of 42 months, cell and animal model systems of defined oncogenes were generated. Exploitation of generated knowledge and tools enabled the consortium to achieve the following research objectives: (1) elucidation of tumor components which confer sensitivity or resistance to TRAIL-induced cell death; (2) providing detailed knowledge on how small molecule Hsp90, Aurora, choline kinase, BRAF inhibitors, DNA damaging agents, HDAC and DNMT inhibitors affect the intrinsic apoptotic amplification and execution machineries; (3) optimization of combined action of TRAIL with these therapeutics for optimum effects with minimum concentrations and toxicity in vivo. These findings provide mechanistic basis for a pharmacogenomic approach, which could be exploited further therapeutically, in order to reach novel personalized therapies for cancer patients. © 2012 Landes Bioscience.This work is supported by the EU grant LSHC-CT-2006-037278 >ONCODEATH>.Peer Reviewe

EZH2 Regulates Cofilin Activity and Colon Cancer Cell Migration by Targeting ITGA2 Gene

<div><p>Reorganization of cytoskeleton via actin remodeling is a basic step of cell locomotion. Although cell migration of normal and cancer cells can be stimulated by a variety of intra- and extra-cellular factors, all paths ultimate on the regulation of cofilin activity. Cofilin is a small actin-binding protein able to bind both forms of actin, globular and filament, and is regulated by phosphorylation at Serine 3. Following phosphorylation at serine 3 cofilin is inactive, therefore cannot bind actin molecules and cytoskeleton remodeling is impaired. The histone methyltransferase EZH2 is frequently over expressed in many tumour types including colorectal cancer (CRC). EZH2 over activity, which results in epigenetic gene-silencing, has been associated with many tumour properties including invasion, angiogenesis and metastasis but little is known about the underneath molecular mechanisms. Herein, we report that EZH2 is able to control cofilin activity and consequently cell locomotion of CRC cell lines through a non-conventional novel axis that involves integrin signaling. Indeed, we show how genetic and pharmacological inhibition (DZNep and GSK343) of EZH2 function produces hyper phosphorylation of cofilin and reduces cell migration. We previously demonstrated by chromatin immuno-precipitation that Integrin alpha 2 (ITGα2) expression is regulated by EZH2. In the present study we provide evidence that in EZH2-silenced cells the signaling activity of the de-repressed ITGα2 is able to increase cofilin phosphorylation, which in turn reduces cell migration. This study also proposes novel mechanisms that might provide new anti-metastatic strategies for CRC treatment based on the inhibition of the epigenetic factor EZH2 and/or its target gene.</p></div

Oncogenic RAS alters the global and gene-specific histone modification pattern during epithelial-mesenchymal transition in colorectal carcinoma cells

The presence of different forms of histone covalent modifications, such as phosphorylation, acetylation and methylation in localized promoter regions are markers for chromatin packing and transcription. Activation of RAS signalling pathways through oncogenic RAS mutations is a hallmark of colorectal cancer. Overexpression of Harvey-Ras oncogene induces epithelial-mesenchymal transition (EMT) in Caco-2 cells. We focused on the role of epigenetic modifications of histone H3 and its dependence on RAS signal transduction pathways and oncogenic transformation. Using cell lines stably overexpressing oncogenic Harvey-RAS with EMT phenotype, we studied the acquired changes in the H3 histone modification patterns. Two genes show inverse protein expression patterns after Ha-RAS overexpression: Cyclin D1, a cell cycle-related gene, and the EMT marker-gene E-cadherin. We report that these two genes demonstrate matching inverse histone repression patterns on their promoter, while histone markers associated with an active state of genes were affected by the RAS-activated signalling pathway MEK-ERK-MSK1. Furthermore, we show that though the level of methyltransferases enzymes was increased, the status of H3 three-methylation at lysine 27 (H3K27me(3)), associated with gene repression on the promoter of Cyclin D1, was lower. Together, these results suggest that histone covalent modifications can be affected by oncogenic RAS pathways to regulate the expression of target genes like Cyclin D1 or E-cadherin and that the dynamic balance of opposing histone-modifying enzymes is critical for the regulation of cell proliferation

EZH2/ΙΤGα2-regulated cofilin activity controls cell morphology and migration.

<p>A. F-actin was stained with phalloin conjugated to a fluorophore (red) in HCT116 and HCTshEZ-2 cells cultured in matrigel. Representative phase contrast microscopy pictures of the same cultures are also presented for each cell line. Arrows indicate long F-actin molecules. Scale bar 20 µm. B. Analysis of relative amount of F- and G-actin in CRC cells with high (HCT116 and HCT-shpSUP) and silenced (HCTsh-EZ-2a/b) EZH2 protein levels. Intensity of WB bands has been quantified and the ratio is calculated in arbitrary units. Representative blot of F- and G-actin is also shown for each cell line. Student T-test was used to evaluate the statistic significance (*** = p-value<0,01) between HCTsh-EZ-2a/b cells and HCT116 cells. C. Migration ability of CRC cell lines transfected with siITGα2. D. WB analysis of p-cofilin levels in CRC cells with high (HCT116 and HCT-shpSUP) and silenced (HCTsh-EZ-2) EZH2 protein treated with ROCK1 inhibitor (Y27632) compared to the untreated cells. E. Migration ability of CRC cell lines treated with ROCK1 inhibitor (Y27632) compared to the untreated cells.</p

P-cofilin, cofilin and G-actin intracellular localization and regulation.

<p>A. HCT116, RKO, HCT-shEZ-2 and HT29 have been co-stained with p-cofilin (green) and G-actin (red) in order to study the intracellular localization of both proteins. Nuclei are counterstained with hoechst (blue). A merge of all three colors is also shown (merge). Scale bars 10 µm. B. Cofilin (green) staining in CRC cells. Only HT29 cells presented almost exclusive cytoplasmic localization as shown by single-plane confocal picture. Scale bars 50 µm. C. Pharmacological disruption of EZH2 protein by DZNep in CRC cell-lines with high EZH2 protein expression. D. Pharmacological inhibition of EZH2 enzymatic activity by GSK343. Low levels of H3K27me3 in treated cells indicate that GSK343 efficiently blocks EZH2 activity. E. TESK1 protein (top) and mRNA (bottom) expression analyses on CRC cells with high (HCT116 and HCT-shpSUP) and silenced (HCTsh-EZ-2a/b) EZH2 protein levels. F. siRNAs versus ITGα2 mRNA have been used to reduce ITGα2 protein expression. The effects of ITGα2 silencing on cofilin and p-cofilin were analyzed using WB. Numbers below blots indicate band quantification which was performed using a dedicated software and Tubulin expression as reference loading control. For p-cofilin quantification in HCT116 and HCT-shEZ-2 cells all values have been normalized with respect to the p-cofilin value of untransfected HCT116. All WB experiments have been repeated at least two times and representative blots are shown.</p

Molecular mechanisms involved in CD43-mediated apoptosis of TF-1 cells - Roles of transcription, Daxx expression, and adhesion molecules

Journal URL: http://www.jbc.org