in-vitro-methylation-assay-to-study-protein-arginine-methylation

<em>In vitro </em>Methylation Assay to Study Protein Arginine Methylation

Protein arginine methylation is one of the most abundant post-translational modifications in the nucleus. Protein arginine methylation can be identified ...

The Soft Agar Colony Formation Assay

Anchorage-independent growth is the ability of transformed cells to grow independently of a solid surface, and is a hallmark of carcinogenesis. The soft ...

University of Illinois at Chicago

Our previous studies revealed that a subset of mammary ductal carcinoma in situ (DCIS) contained focally disrupted myoepithelial (ME) cell layers that were predominantly overlain by estrogen receptor (ER) negative cells, which showed a substantially higher rate of cell proliferation and genetic alterations than adjacent ER positive cells within the same duct. This study attempted to assess whether these cells also had a different expression profile on tumor progression related genes.

cDNA expression profiling reveals elevated gene expression in cell clusters overlying focally disrupted myoepithelial cell layers: implications for breast tumor invasion.

Arachidonic acid (AA) metabolizing enzymes and peroxisome proliferator-activated receptors (PPARs) have been shown to regulate the growth of epithelial cells. We have previously reported that exposure to the 5-lipoxygenase activating protein-directed inhibitor MK886 but not the cyclooxygenase inhibitor, indomethacin, reduced growth, increased apoptosis, and up-regulated PPARalpha and gamma expression in breast cancer cell lines. In the present study, we explore approaches to maximizing the proapoptotic effects of PPARgamma on lung cancer cell lines. Non-small-cell cancer cell line A549 revealed dose-dependent PPARgamma reporter activity after treatment with MK886. The addition of indomethacin in combination with MK886 further increases reporter activity. We also show increased growth inhibition and up-regulation of apoptosis after exposure to MK886 alone, or in combination with indomethacin and the PPAR ligand, 15-deoxy-Delta12,14-prostaglandin J2 compared with single drug exposures on the adenocarcinoma cell line A549 and small-cell cancer cell lines H345, N417, and H510. Real-time PCR analyses showed increased PPAR mRNA and retinoid X receptor (RXR)alpha mRNA expression after exposure to MK886 and indomethacin in a time-dependent fashion. The results suggest that the principal proapoptotic effect of these drugs may be mediated through the known antiproliferative effects of the PPARgamma-RXR interaction. We therefore explored a three-drug approach to attempt to maximize this effect. The combination of low-dose MK886, ciglitazone, and 13-cis-retinoic acid interacted at least in a superadditive fashion to inhibit the growth of lung cancer cell lines A549 and H1299, suggesting that targeting PPARgamma and AA action is a promising approach to lung cancer growth with a favorable therapeutic index.

Inhibitors of the arachidonic acid pathway and peroxisome proliferator-activated receptor ligands have superadditive effects on lung cancer growth inhibition.

Lung cancer is the leading cause of cancer death in the United States and five-year survival remains low. Numerous studies have shown that chronic inflammation may lead to progression of carcinogenesis. As a result of inflammatory stimulation, arachidonic acid (AA) metabolism produces proliferation mediators through complex and dynamic interactions of the products of the LOX/COX enzymes. One important mediator in the activation of the AA pathways is the nuclear protein PPARgamma. Targeting LOX/COX enzymes and inducing activation of PPARgamma have resulted in significant reduction of cell growth in lung cancer cell lines. However, specific COX-inhibitors have been correlated with an increased cardiovascular risk. Clinical applications are still being explored with a novel generation of dual LOX/COX inhibitors. PPARgamma activation through synthetic ligands (TZDs) has revealed a great mechanistic complexity since effects are produced through PPARgamma-dependent and -independent mechanisms. Furthermore, PPARgamma could also be involved in regulation of COX-2. Overexpression of PPARgamma has reported to play a role in control of invasion and differentiation. Exploring the function of PPARgamma, in this new context, may provide a better mechanistic model of its role in cancer and give an opportunity to design a more efficient therapeutic approach in combination with LOX/COX inhibitors.

Combination Therapy of PPARgamma Ligands and Inhibitors of Arachidonic Acid in Lung Cancer.

The association between chronic inflammation and lung cancer has been extensively reported. Despite the differences in the clinical manifestations of chronic obstructive pulmonary disease (COPD) compared with lung cancer, the underlying inflammatory pathogenesis of these two diseases may involve shared biological features. Identifying the mechanisms that regulate specific inflammatory mediators shared by both diseases will represent a major breakthrough. Chemokines play an important role in the inflammatory response. Recent evidence suggests that post-transcriptional regulation, through control of mRNA stability, could be an important mechanism of regulation of chemokine expression. Therefore, it would be important to study the role of RNA-binding proteins, such as hnRNPs, in chemokine expression after initial inflammatory response. HnRNPs have shown the ability to modulate inflammatory mediators expression by affecting mRNA stability of COX-2, TNFalpha and IL-1beta and iNOS. Moreover, overexpression of hnRNPs has been reported in cancer, for example, hnRNP A2/B1 in lung cancer. Thus, investigating the role of hnRNPs in chemokine expression may allow us to understand the molecular mechanisms involved in the regulation of the inflammatory response before its progression to chronic inflammation and/or tumor development.

Lung cancer and inflammation: interaction of chemokines and hnRNPs.

In this study, we investigated the role of hyaluronan (HA) in non-small cell lung cancer (NSCLC) since close association between HA level and malignancy has been reported. HA is an abundant extracellular matrix component and its synthesis is regulated by growth factors and cytokines that include epidermal growth factor (EGF) and interleukin-1beta (IL-1beta). We showed that treatment with recombinant EGF and IL-1beta, alone or in combination with TGF-beta, was able to stimulate HA production in lung adenocarcinoma cell line A549. TGF-beta/IL-1beta treatment induced epithelial to mesenchymal-like phenotype transition (EMT), changing cell morphology and expression of vimentin and E-cadherin. We also overexpressed hyaluronan synthase-3 (HAS3) in epithelial lung adenocarcinoma cell line H358, resulting in induced HA expression, EMT phenotype, enhanced MMP9 and MMP2 activities and increased invasion. Furthermore, adding exogenous HA to A549 cells and inducing HA H358 cells resulted in increased resistance to epidermal growth factor receptor (EGFR) inhibitor, Iressa. Together, these results suggest that elevated HA production is able to induce EMT and increase resistance to Iressa in NSCLC. Therefore, regulation of HA level in NSCLC may be a new target for therapeutic intervention.

Cytokines and growth factors stimulate hyaluronan production: role of hyaluronan in epithelial to mesenchymal-like transition in non-small cell lung cancer.

Heterogeneous nuclear ribonucleoprotein A2/B1 (hnRNP A2/B1) has been reported to be overexpressed in lung cancer and in other cancers such as breast, pancreas, and liver. However, a mechanism linking hnRNP A2/B1 overexpression and progression to cancer has not yet been definitively established. To elucidate this mechanism, we have silenced hnRNPA2/B1 mRNA in non-small-cell lung cancer cell lines A549, H1703, and H358. These cell lines present different levels of expression of epithelial-to-mesenchymal transition (EMT) markers such as E-cadherin, fibronectin, and vimentin. Microarray expression analysis was performed to evaluate the effect of silencing hnRNP A2/B1 in A549 cells. We identified a list of target genes, affected by silencing of hnRNP A2/B1, that are involved in regulation of migration, proliferation, survival, and apoptosis. Silencing hnRNP A2/B1 induced formation of cell clusters and increased proliferation. In the anchorage-independent assay, silencing hnRNP A2/B1 increased colony formation by 794% in A549 and 174% in H1703 compared with a 25% increase in proliferation, in both cell lines, in a two-dimensional proliferation assay. Silencing hnRNP A2/B1 decreased migration in intermediate cell line A549 and mesenchymal cell line H1703; however, no changes in proliferation were observed in epithelial cell line H358. Silencing hnRNP A2/B1 in A549 and H1703 cells correlated with an increase of E-cadherin expression and downregulation of the E-cadherin inhibitors Twist1 and Snai1. These data suggest that expression of hnRNP A2/B1 may play a role in EMT, in nonepithelial lung cancer cell lines A549 and H1703, through the regulation of E-cadherin expression.

hnRNP A2/B1 modulates epithelial-mesenchymal transition in lung cancer cell lines.

Heparanase-1 (HPR1), an endoglycosidase that specifically degrades heparan sulfate (HS) proteoglycans, is overexpressed in a variety of malignancies. Our present study sought to determine whether oncogene BRAF and RAS mutations lead to increased HPR1 expression. Reverse transcription-polymerase chain reaction analysis revealed that HPR1 gene expression was increased in HEK293 cells transiently transfected with a mutant BRAF or RAS gene. Flow cytometric analysis revealed that B-Raf activation led to loss of the cell surface HS, which could be blocked by two HPR1 inhibitors: heparin and PI-88. Cotransfection of a BRAF or RAS mutant gene with HPR1 promoter-driven luciferase reporters increased luciferase reporter gene expression in HEK293 cells. Knockdown of BRAF expression in a BRAF-mutated KAT-10 tumor cell line led to the suppression of HPR1 gene expression, subsequently leading to increased cell surface HS levels. Truncational and mutational analyses of the HPR1 promoter revealed that the Ets-relevant elements in the HPR1 promoter were critical for BRAF activation-induced HPR1 expression. Luciferase reporter gene expression driven by a four-copy GA binding protein (GABP) binding site was significantly lower in BRAF siRNA-transfected KAT-10 cells than in the control siRNA-transfected cells. We further showed that BRAF knockdown led to suppression of the expression of the GABPβ, an Ets family transcription factor involved in regulating HPR1 promoter activity. Taken together, our study suggests that B-Raf kinase activation plays an important role in regulating HPR1 expression. Increased HPR1 expression may contribute to the aggressive behavior of BRAF-mutated cancer.

ArticleTotal : 2	Year
Publication title Cited by 8	2014
Publication title Cited by 525	2014

Article	Year
cDNA expression profiling reveals elevated gene expression in cell clusters overlying focally disrupted myoepithelial cell layers: implications for breast tumor invasion. Breast cancer research and treatment\| PubMed ID: 15692763	2005
Inhibitors of the arachidonic acid pathway and peroxisome proliferator-activated receptor ligands have superadditive effects on lung cancer growth inhibition. Cancer research\| PubMed ID: 15899809	2005
Combination Therapy of PPARgamma Ligands and Inhibitors of Arachidonic Acid in Lung Cancer. PPAR research\| PubMed ID: 19277204	2008
Lung cancer and inflammation: interaction of chemokines and hnRNPs. Current opinion in pharmacology\| PubMed ID: 19570713	2009
Cytokines and growth factors stimulate hyaluronan production: role of hyaluronan in epithelial to mesenchymal-like transition in non-small cell lung cancer. Journal of biomedicine & biotechnology\| PubMed ID: 20671927	2010
hnRNP A2/B1 modulates epithelial-mesenchymal transition in lung cancer cell lines. Cancer research\| PubMed ID: 20807810	2010
Induction of heparanase-1 expression by mutant B-Raf kinase: role of GA binding protein in heparanase-1 promoter activation. Neoplasia (New York, N.Y.)\| PubMed ID: 21076620	2010

Jordi Tauler

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