u-0126 and Neoplasms

Cancer-associated cachexia (CAC) is a multifactorial wasting syndrome characterized by loss of skeletal muscle. Interleukin-11 (IL11), one of the IL6 family cytokines, is highly expressed in various types of cancer including cancers frequently associated with cachexia. However, the impact of IL11 on muscle metabolism remains to be determined. Since one of the mechanisms of muscle wasting in cachexia is defective muscle regeneration due to impaired myogenic differentiation, we examined the effect of IL11 on the differentiation of C2C12 mouse myoblasts. Treatment of C2C12 cells with recombinant mouse IL11 resulted in decreased myotube formation. In addition, IL11 treatment reduced the protein and mRNA levels of myosin heavy chain (MHC), a marker of myogenic differentiation. Moreover, the levels of myogenic regulatory factors including myogenin and Mrf4 were significantly reduced by IL11 treatment. IL11 treatment increased the number of BrdU-positive cells and the level of phosphorylated retinoblastoma (Rb) protein, while the levels of p21

Topics: Animals; Bromodeoxyuridine; Cachexia; Cell Differentiation; Extracellular Signal-Regulated MAP Kinases; Interleukin-11; Mice; Muscle Development; Myoblasts; Myogenin; Myosin Heavy Chains; Neoplasms

Cancer cells generally exhibit increased iron uptake, which contributes to their abnormal growth and metastatic ability. Iron chelators have thus recently attracted attention as potential anticancer agents. Here, we show that deferriferrichrysin (Dfcy), a natural product from Aspergillus oryzae acts as an iron chelator to induce paraptosis (a programmed cell death pathway characterized by ER dilation) in MCF-7 human breast cancer cells and H1299 human lung cancer cells. We first examined the anticancer efficacy of Dfcy in cancer cells and found that Dfcy induced ER dilation and reduced the number of viable cells. Extracellular signal-related kinase (ERK) was activated by Dfcy treatment, and the MEK inhibitor U0126, a small molecule commonly used to inhibit ERK activity, prevented the increase in ER dilation in Dfcy-treated cells. Concomitantly, the decrease in the number of viable cells upon treatment with Dfcy was attenuated by U0126. Taken together, these results demonstrate that the iron chelator Dfcy exhibits anticancer effects via induction of ERK-dependent paraptosis.

Topics: Apoptosis; Cell Line, Tumor; Extracellular Signal-Regulated MAP Kinases; Humans; Iron Chelating Agents; Neoplasms

Oncolytic viruses are potent anticancer agents that replicate within and kill cancer cells rather than normal cells, and their selectivity is largely determined by oncogenic mutations. M1, a novel oncolytic virus strain, has been shown to target cancer cells, but the relationship between its cancer selectivity and oncogenic signaling pathways is poorly understood. Here, we report that RAS mutation promotes the replication and oncolytic effect of M1 in cancer, and we further provide evidence that the inhibition of the RAS/RAF/MEK signaling axis suppresses M1 infection and the subsequent cytopathic effects. Transcriptome analysis revealed that the inhibition of RAS signaling upregulates the type I interferon antiviral response, and further RNA interference screen identified CDKN1A as a key downstream factor that inhibits viral infection. Gain- and loss-of-function experiments confirmed that CDKN1A inhibited the replication and oncolytic effect of M1 virus. Subsequent TCGA data mining and tissue microarray (TMA) analysis revealed that CDKN1A is commonly deficient in human cancers, suggesting extensive clinical application prospects for M1. Our report indicates that virotherapy is feasible for treating undruggable RAS-driven cancers and provides reliable biomarkers for personalized cancer therapy.

Topics: Animals; Antiviral Agents; Biomarkers, Tumor; Butadienes; Cell Line; Cell Line, Tumor; Cyclin-Dependent Kinase Inhibitor p21; Female; Gene Expression Regulation, Viral; Humans; Mice, Inbred BALB C; Mice, Nude; Mutation; Neoplasms; Nitriles; Oncolytic Viruses; ras Proteins; RNA, Small Interfering; Signal Transduction; Up-Regulation; Virus Replication

Resveratrol, a natural polyphenol compound, has been shown to possess anticancer activity. However, how resveratrol inhibits cancer cell adhesion has not been fully elucidated. Here, we show that resveratrol suppressed the basal or type I insulin-like growth factor (IGF)-1-stimulated adhesion of cancer cells (Rh1, Rh30, HT29, and HeLa cells) by inhibiting the extracellular signal-regulated kinase 1/2 (Erk1/2) pathway. Inhibition of Erk1/2 with U0126, knockdown of Erk1/2, or overexpression of dominant-negative mitogen-activated protein kinase kinase 1 (MKK1) strengthened resveratrol's inhibition of the basal or IGF-1-stimulated of Erk1/2 phosphorylation and cell adhesion, whereas ectopic expression of constitutively active MKK1 attenuated the inhibitory effects of resveratrol. Further research revealed that both protein phosphatase 2A (PP2A) and phosphatase and tensin homolog (PTEN)-Akt were implicated in resveratrol-inactivated Erk1/2-dependent cell adhesion. Inhibition of PP2A with okadaic acid or overexpression of dominant-negative PP2A rendered resistance to resveratrol's suppression of the basal or IGF-1-stimulated phospho-Erk1/2 and cell adhesion, whereas expression of wild-type PP2A enhanced resveratrol's inhibitory effects. Overexpression of wild-type PTEN or dominant-negative Akt or inhibition of Akt with Akt inhibitor X strengthened resveratrol's inhibition of the basal or IGF-1-stimulated Erk1/2 phosphorylation and cell adhesion. Furthermore, inhibition of mechanistic/mammalian target of rapamycin (mTOR) with rapamycin or silencing mTOR enhanced resveratrol's inhibitory effects on the basal and IGF-1-induced inhibition of PP2A-PTEN, activation of Akt-Erk1/2, and cell adhesion. The results indicate that resveratrol inhibits Erk1/2-mediated adhesion of cancer cells via activating PP2A-PTEN signaling network. Our data highlight that resveratrol has a great potential in the prevention of cancer cell adhesion.

Topics: Butadienes; Cell Adhesion; Drug Resistance, Neoplasm; Gene Expression Regulation, Neoplastic; HeLa Cells; HT29 Cells; Humans; Insulin-Like Growth Factor I; MAP Kinase Kinase 1; MAP Kinase Signaling System; Neoplasms; Nitriles; Okadaic Acid; Oncogene Protein v-akt; Protein Phosphatase 2; PTEN Phosphohydrolase; Resveratrol; Signal Transduction; TOR Serine-Threonine Kinases

Cancer cell migration requires that cells respond and adapt to their surroundings. In the absence of extracellular matrix cues, cancer cells will undergo a mesenchymal to ameboid transition, whereas a highly confining space will trigger a switch to "leader bleb-based" migration. To identify oncogenic signaling pathways mediating these transitions, we undertook a targeted screen using clinically useful inhibitors. Elevated Src activity was found to change actin and focal adhesion dynamics, whereas inhibiting Src triggered focal adhesion disassembly and blebbing. On non-adherent substrates and in collagen matrices, amoeboid-like, blebbing cells having high Src activity formed protrusions of the plasma membrane. To evaluate the role of Src in confined cells, we use a novel approach that places cells under a slab of polydimethylsiloxane (PDMS), which is held at a defined height. Using this method, we find that leader bleb-based migration is resistant to Src inhibition. High Src activity was found to markedly change the architecture of cortical actomyosin, reduce cell mechanical properties, and the percentage of cells that undergo leader bleb-based migration. Thus, Src is a signal transducer that can potently influence transitions between migration modes with implications for the rational development of metastasis inhibitors.

Topics: Actins; Amino Acid Substitution; Butadienes; Cell Adhesion; Cell Movement; Dasatinib; Humans; Imidazoles; Mutant Proteins; Neoplasms; Nitriles; Phosphorylcholine; Protein Kinase Inhibitors; Proto-Oncogene Proteins pp60(c-src); Quinolines; Tumor Cells, Cultured

Mitogen activated protein kinase (MAPK) signal transduction pathway has been proved to play an important role in tumorigenesis and cancer development. MEK inhibitor has been demonstrated significant clinical benefit for blocking MAPK pathway activation and possibly could block reactivation of the MAPK pathway at the time of BRAF inhibitor resistance. Twenty N-(benzyloxy)-1,3-diphenyl-1H-pyrazole-4-carboxamide derivatives have been designed and synthesized as MEK inhibitors, and their biological activities were evaluated. Among these compounds, compound 7b showed the most potent inhibitory activity with IC50 of 91nM for MEK1 and GI50 value of 0.26μM for A549 cells. The SAR analysis and docking simulation were performed to provide crucial pharmacophore clues that could be used in further structure optimization.

Topics: Antineoplastic Agents; Biphenyl Compounds; Cell Line, Tumor; Cell Proliferation; Humans; MAP Kinase Kinase 1; MAP Kinase Signaling System; Molecular Docking Simulation; Neoplasms; Protein Kinase Inhibitors; Pyrazoles; Quantitative Structure-Activity Relationship

The p53 tumor suppressor inhibits cell growth through the activation of both cell cycle arrest and apoptosis, which maintain genome stability and prevent cancer development. Here, we report that intercellular adhesion molecule-2 (ICAM2) is transcriptionally activated by p53. Specifically, ICAM2 is induced by the p53 family and DNA damage in a p53-dependent manner. We identified a p53 binding sequence located within the ICAM2 gene that is responsive to wild-type p53, TAp73, and TAp63. In terms of function, we found that the ectopic expression of ICAM2 inhibited cancer cell migration and invasion. In addition, we demonstrated that silencing endogenous ICAM2 in cancer cells caused a marked increase in extracellular signal-regulated kinase (ERK) phosphorylation levels, suggesting that ICAM2 inhibits migration and invasion of cancer cells by suppressing ERK signaling. Moreover, ICAM2 is underexpressed in human cancer tissues containing mutant p53 as compared to those with wild-type p53. Notably, the decreased expression of ICAM2 is associated with poor survival in patients with various cancers. Our findings demonstrate that ICAM2 induction by p53 has a key role in inhibiting migration and invasion.

Topics: Antigens, CD; Apoptosis; Butadienes; Cell Adhesion Molecules; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Movement; Cell Proliferation; Chromatin Immunoprecipitation; DNA Damage; Extracellular Signal-Regulated MAP Kinases; Gene Expression Regulation, Neoplastic; Humans; MAP Kinase Signaling System; Mutation; Neoplasm Invasiveness; Neoplasms; Nitriles; Phosphorylation; RNA Interference; RNA, Small Interfering; Transcription Factors; Transcriptional Activation; Tumor Protein p73; Tumor Suppressor Protein p53; Tumor Suppressor Proteins

Interferon regulatory factor (IRF1) is a potent antiviral, antitumor and immune regulatory protein. Recently, we found that activated Ras/MEK inhibits antiviral response by downregulating IRF1 expression and renders cancer cells susceptible to oncolytic viruses. In this study, we sought to determine whether IRF1 downregulation underlies oncogenesis induced by Ras/MEK activation in human cancer cells. Treatment of the MEK inhibitor U0126 promoted IRF1 expression in 7 of 11 cancer cell lines we tested. IRF1 promotion was also observed in human cancer cell lines treated with different MEK inhibitors or with RNAi oligonucleotides against extracellular signal-regulated kinases (ERKs). Restoration of the expression of antitumor genes, p27 and p53 upregulated modulator of apoptosis (PUMA), by MEK inhibition was less in IRF1 shRNA knockdown cancer cells than in vector control cancer cells, suggesting that Ras/MEK targets IRF1 for the downregulation of the antitumor genes. Moreover, apoptosis induction by U0126 was significantly reduced in IRF1 shRNA knockdown cells than vector control cells. This study demonstrates that IRF1 expression is suppressed by activated Ras/MEK in human cancer cells and that IRF1 plays essential roles in apoptosis induced by Ras/MEK inhibition.

Topics: Animals; Apoptosis; Apoptosis Regulatory Proteins; Blotting, Western; Butadienes; Cell Line, Tumor; Cell Survival; Cyclin-Dependent Kinase Inhibitor p27; Dose-Response Relationship, Drug; Enzyme Inhibitors; Gene Expression Regulation, Neoplastic; HCT116 Cells; HT29 Cells; Humans; Interferon Regulatory Factor-1; MCF-7 Cells; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Models, Genetic; Neoplasms; Nitriles; Proto-Oncogene Proteins; ras Proteins; Reverse Transcriptase Polymerase Chain Reaction; RNA Interference; Signal Transduction; Tumor Suppressor Protein p53

At the initial stage of carcinogenesis, a mutation occurs in a single cell within a normal epithelial layer. We have previously shown that RasV12-transformed cells are apically extruded from the epithelium when surrounded by normal cells. However, the molecular mechanisms underlying this phenomenon remain elusive. Here, we demonstrate that Cav-1-containing microdomains and EPLIN (also known as LIMA1) are accumulated in RasV12-transformed cells that are surrounded by normal cells. We also show that knockdown of Cav-1 or EPLIN suppresses apical extrusion of RasV12-transformed cells, suggesting their positive role in the elimination of transformed cells from epithelia. EPLIN functions upstream of Cav-1 and affects its enrichment in RasV12-transformed cells that are surrounded by normal cells. Furthermore, EPLIN regulates non-cell-autonomous activation of myosin-II and protein kinase A (PKA) in RasV12-transformed cells. In addition, EPLIN substantially affects the accumulation of filamin A, a vital player in epithelial defense against cancer (EDAC), in the neighboring normal cells, and vice versa. These results indicate that EPLIN is a crucial regulator of the interaction between normal and transformed epithelial cells.

Topics: Animals; Butadienes; Caveolae; Caveolin 1; Cell Line; Cell Transformation, Neoplastic; Chromones; Contactin 1; Cyclic AMP-Dependent Protein Kinases; Dogs; Enzyme Inhibitors; Epithelial Cells; Extracellular Signal-Regulated MAP Kinases; Filamins; Madin Darby Canine Kidney Cells; MAP Kinase Signaling System; Microfilament Proteins; Morpholines; Myosin Type II; Neoplasms; Nitriles; Phosphoinositide-3 Kinase Inhibitors; Proto-Oncogene Proteins p21(ras); RNA Interference; RNA, Small Interfering

Although clinical studies have evaluated several MEK1/2 inhibitors, it is unlikely that MEK1/2 inhibitors will be studied clinically. BRAF mutations have been proposed as a responder marker of MEK1/2 inhibitors in a preclinical study. However, current clinical approaches focusing on BRAF mutations have shown only moderate sensitivity of MEK1/2 inhibitors. This has led to insufficient support for their promoted clinical adoption. Further characterization of tumors sensitive to MEK inhibitors holds great promise for optimizing drug therapy for patients with these tumors. Here, we report that β-catenin mutations accelerate apoptosis induced by MEK1/2 inhibitor. SMK-17, a selective MEK1/2 inhibitor, induced apoptosis in tumor cell lines harboring β-catenin mutations at its effective concentration. To confirm that β-catenin mutations and mutant β-catenin-mediated TCF7L2 (also known as TCF4) transcriptional activity is a predictive marker of MEK inhibitors, we evaluated the effects of dominant-negative TCF7L2 and of active, mutated β-catenin on apoptosis induced by MEK inhibitor. Indeed, dominant-negative TCF7L2 reduced apoptosis induced by MEK inhibitor, whereas active, mutated β-catenin accelerated it. Our findings show that β-catenin mutations are an important responder biomarker for MEK1/2 inhibitors.

Topics: Animals; Antineoplastic Agents; Apoptosis; beta Catenin; Butadienes; Cell Line, Tumor; Cell Proliferation; Diphenylamine; Humans; MAP Kinase Kinase 1; MAP Kinase Kinase 2; Mice; Mice, Inbred NOD; Mice, SCID; Mutation; Neoplasms; Nitriles; Protein Kinase Inhibitors; Sulfonamides; Transcription Factor 7-Like 2 Protein; Transcription, Genetic

Dysregulated MAPK/ERK signaling is implicated in one-third of human tumors and represents an attractive target for the development of anticancer drugs. Similarly, elevated protein O-GlcNAcylation and O-GlcNAc transferase (OGT) are detected in various cancers and serve as attractive novel cancer-specific therapeutic targets. However, the potential connection between them remains unexplored. Here, a positive correlation was found between the activated MAPK/ERK signaling and hyper-O-GlcNAcylation in various cancer types and inhibition of the MAPK/ERK signaling by 10 µM U0126 significantly decreased the expression of OGT and O-GlcNAcylation in H1299, BPH-1 and DU145 cells; then, the pathway analysis of the potential regulators of OGT obtained from the UCSC Genome Browser was done, and ten downstream targets of ERK pathway were uncovered; the following results showed that ELK1, one of the ten targets of ERK pathway, mediated ERK signaling-induced OGT upregulation; finally, the MTT assay and the soft agar assay showed that the inhibition of MAPK/ERK signaling reduced the promotion effect of hyper-O-GlcNAcylation on cancer cell proliferation and anchorage-independent growth. Taken together, our data originally provided evidence for the regulatory mechanism of hyper-O-GlcNAcylation in tumors, which will be helpful for the development of anticancer drugs targeting to hyper-O-GlcNAcylation. This study also provided a new mechanism by which MAPK/ERK signaling-enhanced cancer malignancy. Altogether, the recently discovered oncogenic factor O-GlcNAc was linked to the classical MAPK/ERK signaling which is essential for the maintenance of malignant phenotype of cancers.

Topics: Antineoplastic Agents; Butadienes; Cell Line, Tumor; Cell Proliferation; Enzyme Activation; ets-Domain Protein Elk-1; Extracellular Signal-Regulated MAP Kinases; Glycosylation; Humans; MAP Kinase Signaling System; N-Acetylglucosaminyltransferases; Neoplasms; Nitriles; Protein Kinase Inhibitors; Protein Processing, Post-Translational; RNA Interference; Transfection

Granulocyte colony-stimulating factor (G-CSF) affects granulopoiesis and is important for mobilizing neutrophils into blood circulation. Due to the hematopoietic properties of G-CSF, it has been widely used to clinically treat chemotherapy-induced neutropenia. However, G-CSF can promote tumors by inhibiting innate and adaptive immunity and enhancing angiogenesis and neoplastic growth. Most G-CSF-producing tumors are associated with a poor prognosis. This indicates that G-CSF promotes cancer progression. Thus, identifying regulatory molecules involved in tumor-derived G-CSF expression may provide therapeutic targets for cancer treatment. This study identified considerable G-CSF expression in malignant breast, lung and oral cancer cells. However, G-CSF expression was barely detectable in non-invasive cell lines. Expression of G-CSF mRNA and protein increased during exposure to tumor necrosis factor-α (TNF-α). Treatment with U0126 (a mitogen-activated protein kinase inhibitor) drastically reduced basal levels of G-CSF and TNF-α-induced G-CSF in aggressive cancer cells. This study also showed that knockdown of extracellular signal-regulated kinase (ERK) 2 by shRNA was necessary and sufficient to eliminate the expression of tumor-derived G-CSF. This did not apply to ERK1. Therefore, ERK2 (but not ERK1) is responsible for the transcriptional regulation of tumor-derived G-CSF. The results indicate the pharmaceutical value of specific ERK2 inhibitors in treating patients with G-CSF-producing tumors.

Topics: Adenocarcinoma; Adenocarcinoma of Lung; Breast Neoplasms; Butadienes; Carcinoma, Squamous Cell; Cell Line, Tumor; Enzyme Inhibitors; Female; Gene Expression Regulation, Neoplastic; Granulocyte Colony-Stimulating Factor; Humans; Lung Neoplasms; MAP Kinase Signaling System; Mitogen-Activated Protein Kinase 1; Mouth Neoplasms; Neoplasm Invasiveness; Neoplasms; Nitriles; RNA Interference; RNA, Messenger; RNA, Small Interfering; Tumor Necrosis Factor-alpha

Protein-protein interactions mediated through the C-terminal Bcl-2-associated athanogene (BAG) domain of BAG-1 are critical for cell survival and proliferation. Thioflavin S (NSC71948)-a mixture of compounds resulting from the methylation and sulfonation of primulin base-has been shown to dose-dependently inhibit the interaction between BAG-1 and Hsc70 in vitro. In human breast cancer cell lines, with high BAG-1 expression levels, Thioflavin S reduces the binding of BAG-1 to Hsc70, Hsp70, or CRAF and decreases proliferation and viability. Here, we report the development of a protocol for the purification and isolation of biologically active constituents of Thioflavin S and the characterization of the novel compound Thio-2. Thio-2 blocked the growth of several transformed cell lines, but had much weaker effects on untransformed cells. Thio-2 also inhibited the proliferation of melanoma cell lines that had become resistant to treatment with PLX4032, an inhibitor of mutant BRAF. In transformed cells, Thio-2 interfered with intracellular signaling at the level of RAF, but had no effect on the activation of AKT. Thio-2 decreased binding of BAG-1 to Hsc70 and to a lesser extent BRAF in vitro and in vivo, suggesting a possible mechanism of action. Given that tumors frequently develop resistance to kinase inhibitors during treatment, Thio-2 and related compounds may offer promising alternative strategies to currently available therapies.

Topics: Aniline Compounds; Animals; Antineoplastic Agents; Benzothiazoles; Binding Sites; Butadienes; Cell Line; Cell Proliferation; DNA-Binding Proteins; Fluorescent Dyes; HEK293 Cells; HSC70 Heat-Shock Proteins; Humans; Indoles; MCF-7 Cells; Mice; Molecular Docking Simulation; Neoplasms; NIH 3T3 Cells; Nitriles; Protein Binding; Proto-Oncogene Proteins B-raf; Sulfonamides; Thiazoles; Transcription Factors; Vemurafenib

CIB1 is a 22-kDa regulatory protein previously implicated in cell survival and proliferation. However, the mechanism by which CIB1 regulates these processes is poorly defined. Here, we report that CIB1 depletion in SK-N-SH neuroblastoma and MDA-MB-468 breast cancer cells promotes non-apoptotic, caspase-independent cell death that is not initiated by increased outer mitochondrial membrane permeability or translocation of apoptosis-inducing factor to the nucleus. Instead, cell death requires nuclear GAPDH accumulation. Furthermore, CIB1 depletion disrupts two commonly dysregulated, oncogenic pathways-PI3K/AKT and Ras/MEK/ERK, resulting in a synergistic mechanism of cell death, which was mimicked by simultaneous pharmacological inhibition of both pathways, but not either pathway alone. In defining each pathway's contributions, we found that AKT inhibition alone maximally induced GAPDH nuclear accumulation, whereas MEK/ERK inhibition alone had no effect on GAPDH localization. Concurrent GAPDH nuclear accumulation and ERK inhibition were required, however, to induce a significant DNA damage response, which was critical to subsequent cell death. Collectively, our results indicate that CIB1 is uniquely positioned to regulate PI3K/AKT and MEK/ERK signaling and that simultaneous disruption of these pathways synergistically induces a nuclear GAPDH-dependent cell death. The mechanistic insights into cell death induced by CIB1 interference suggest novel molecular targets for cancer therapy.

Topics: Butadienes; Calcium-Binding Proteins; Cell Cycle; Cell Death; Cell Line, Tumor; Cell Nucleus; Enzyme Inhibitors; Extracellular Signal-Regulated MAP Kinases; Glyceraldehyde-3-Phosphate Dehydrogenase (Phosphorylating); Humans; Immunoblotting; Immunohistochemistry; Iodoacetates; MAP Kinase Signaling System; Mitogen-Activated Protein Kinases; Neoplasms; Nitriles; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Proto-Oncogene Proteins c-akt; ras Proteins; RNA Interference; Selegiline; Signal Transduction

The epidermal growth factor receptor (EGFR) is a well-established target for cancer treatment. EGFR tyrosine kinase (TK) inhibitors, such as gefinitib and erlotinib, have been developed as anti-cancer drugs. Although non-small cell lung carcinoma with an activating EGFR mutation, L858R, responds well to gefinitib and erlotinib, tumors with a doubly mutated EGFR, T790M-L858R, acquire resistance to these drugs. The C. elegans EGFR homolog LET-23 and its downstream signaling pathway have been studied extensively to provide insight into regulatory mechanisms conserved from C. elegans to humans. To develop an in vivo screening system for potential cancer drugs targeting specific EGFR mutants, we expressed three LET-23 chimeras in which the TK domain was replaced with either the human wild-type TK domain (LET-23::hEGFR-TK), a TK domain with the L858R mutation (LET-23::hEGFR-TK[L858R]), or a TK domain with the T790M-L858R mutations (LET-23::hEGFR-TK[T790M-L858R]) in C. elegans vulval cells using the let-23 promoter. The wild-type hEGFR-TK chimeric protein rescued the let-23 mutant phenotype, and the activating mutant hEGFR-TK chimeras induced a multivulva (Muv) phenotype in a wild-type C. elegans background. The anti-cancer drugs gefitinib and erlotinib suppressed the Muv phenotype in LET-23::hEGFR-TK[L858R]-expressing transgenic animals, but not in LET-23::hEGFR-TK[T790M-L858R] transgenic animals. As a pilot screen, 8,960 small chemicals were tested for Muv suppression, and AG1478 (an EGFR-TK inhibitor) and U0126 (a MEK inhibitor) were identified as potential inhibitors of EGFR-mediated biological function. In conclusion, transgenic C. elegans expressing chimeric LET-23::hEGFR-TK proteins are a model system that can be used in mutation-specific screens for new anti-cancer drugs.

Topics: Animals; Antineoplastic Agents; Butadienes; Caenorhabditis elegans; Drug Screening Assays, Antitumor; Enzyme Inhibitors; ErbB Receptors; Humans; Models, Animal; Mutation; Neoplasms; Nitriles; Phenotype; Protein Structure, Tertiary; Quinazolines; Transgenes; Tyrphostins

Certain oncolytic viruses exploit activated Ras signaling in order to replicate in cancer cells. Constitutive activation of the Ras/MEK pathway is known to suppress the effectiveness of the interferon (IFN) antiviral response, which may contribute to Ras-dependent viral oncolysis. Here, we identified 10 human cancer cell lines (out of 16) with increased sensitivity to the anti-viral effects of IFN-α after treatment with the MEK inhibitor U0126, suggesting that the Ras/MEK pathway underlies their reduced sensitivity to IFN. To determine how Ras/MEK suppresses the IFN response in these cells, we used DNA microarrays to compare IFN-induced transcription in IFN-sensitive SKOV3 cells, moderately resistant HT1080 cells, and HT1080 cells treated with U0126. We found that 267 genes were induced by IFN in SKOV3 cells, while only 98 genes were induced in HT1080 cells at the same time point. Furthermore, the expression of a distinct subset of IFN inducible genes, that included RIGI, GBP2, IFIT2, BTN3A3, MAP2, MMP7 and STAT2, was restored or increased in HT1080 cells when the cells were co-treated with U0126 and IFN. Bioinformatic analysis of the biological processes represented by these genes revealed increased representation of genes involved in the anti-viral response, regulation of apoptosis, cell differentiation and metabolism. Furthermore, introduction of constitutively active Ras into IFN sensitive SKOV3 cells reduced their IFN sensitivity and ability to activate IFN-induced transcription. This work demonstrates for the first time that activated Ras/MEK in human cancer cells induces downregulation of a specific subset of IFN-inducible genes.

Topics: Antiviral Agents; Butadienes; Cell Line, Tumor; Cell Transformation, Neoplastic; Drug Resistance, Neoplasm; Enzyme Activation; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Humans; Interferons; Mitogen-Activated Protein Kinase Kinases; Molecular Sequence Annotation; Neoplasms; Nitriles; Oligonucleotide Array Sequence Analysis; ras Proteins; Reproducibility of Results; Reverse Transcriptase Polymerase Chain Reaction; Transcription, Genetic

Protein kinases have important functions in the control of cell biology and are implicated in several diseases including cancer. Here we describe a technique to quantify protein kinase activity in a global fashion and without preconception of the kinases that may be active in the cell or tissue under investigation. In Global Kinase Activity Profiling (GKAP), protein kinases present in experimental cell lysates phosphorylate endogenous substrates, also present in the lysate, under defined conditions. Reaction products are then quantified using standard phosphoproteomic techniques based on LC-MS/MS. The technique thus allows measuring the combined activities of kinases targeting common substrates, which are detected as phosphopeptides by LC-MS/MS. Almost four hundred kinase reactions could be quantified in a human epithelial cell line, 177 of which increased in response to EGF treatment while others decreased in cells exposed to the kinase inhibitors LY294002 or U0126. GKAP also detected marked differences in the patterns of kinase activities in human leukemia cell lines with different sensitivities to kinase inhibitors. These results reveal that GKAP detects and quantifies hundreds of kinase activities modulated by growth factors or pharmacological inhibitors, and that these activities correlate with the phenotypes of cancer cells and their responses to kinase inhibitors.

Topics: Butadienes; Cell Line, Tumor; Chromones; Enzyme Inhibitors; Humans; Morpholines; Neoplasm Proteins; Neoplasms; Nitriles; Protein Kinases; Proteome

A small molecule compound, JTP-74057/GSK1120212/trametinib, had been discovered as a very potent antiproliferative agent able to induce the accumulation of CDK inhibitor p15INK4b. To conduct its drug development rationally as an anticancer agent, molecular targets of this compound were identified as MEK1/2 using compound-affinity chromatography. It was shown that JTP-74057 directly bound to MEK1 and MEK2 and allosterically inhibited their kinase activities, and that its inhibitory characteristics were similar to those of the known and different chemotype of MEK inhibitors PD0325901 and U0126. It was further shown that JTP-74057 induced rapid and sustained dephosphorylation of phosphorylated MEK in HT-29 colon and other cancer cell lines, while this decrease in phosphorylated MEK was not observed in PD0325901-treated cancer cells. Physicochemical analyses revealed that JTP-74057 preferentially binds to unphosphorylated MEK (u-MEK) in unique characteristics of both high affinity based on extremely low dissociation rates and ability stabilizing u-MEK with high thermal shift, which were markedly different from PD0325901. These findings indicate that JTP-74057 is a novel MEK inhibitor able to sustain MEK to be an unphosphorylated form resulting in pronounced suppression of the downstream signaling pathways involved in cellular proliferation.

Topics: Allosteric Regulation; Antineoplastic Agents; Benzamides; Butadienes; Chromatography, Affinity; Diphenylamine; Dose-Response Relationship, Drug; HEK293 Cells; HT29 Cells; Humans; Kinetics; MAP Kinase Kinase 1; MAP Kinase Kinase 2; Molecular Structure; Molecular Targeted Therapy; Neoplasms; Nitriles; Phosphorylation; Protein Binding; Protein Kinase Inhibitors; Pyridones; Pyrimidinones; Signal Transduction

The mammalian target of rapamycin (mTOR) which is part of two functionally distinct complexes, mTORC1 and mTORC2, plays an important role in vascular endothelial cells. Indeed, the inhibition of mTOR with an allosteric inhibitor such as rapamycin reduces the growth of endothelial cell in vitro and inhibits angiogenesis in vivo. Recent studies have shown that blocking mTOR results in the activation of other prosurvival signals such as Akt or MAPK which counteract the growth inhibitory properties of mTOR inhibitors. However, little is known about the interactions between mTOR and MAPK in endothelial cells and their relevance to angiogenesis. Here we found that blocking mTOR with ATP-competitive inhibitors of mTOR or with rapamycin induced the activation of the mitogen-activated protein kinase (MAPK) in endothelial cells. Downregulation of mTORC1 but not mTORC2 had similar effects showing that the inhibition of mTORC1 is responsible for the activation of MAPK. Treatment of endothelial cells with mTOR inhibitors in combination with MAPK inhibitors reduced endothelial cell survival, proliferation, migration and tube formation more significantly than either inhibition alone. Similarly, in a tumor xenograft model, the anti-angiogenic efficacy of mTOR inhibitors was enhanced by the pharmacological blockade of MAPK. Taken together these results show that blocking mTORC1 in endothelial cells activates MAPK and that a combined inhibition of MAPK and mTOR has additive anti-angiogenic effects. They also provide a rationale to target both mTOR and MAPK simultaneously in anti-angiogenic treatment.

Topics: Angiogenesis Inhibitors; Animals; Butadienes; Cell Movement; Cell Proliferation; Cell Survival; Cells, Cultured; Endothelium, Vascular; Enzyme Inhibitors; Humans; Imidazoles; Male; Mechanistic Target of Rapamycin Complex 1; Mice; Mice, Nude; Mitogen-Activated Protein Kinase Kinases; Morpholines; Multiprotein Complexes; Neoplasms; Neovascularization, Pathologic; Nitriles; Proteins; Pyrimidines; Quinolines; Sirolimus; TOR Serine-Threonine Kinases; Transcription Factors

Ten years have passed since the emergence of microarray technology. Recent microarray procedures have provided reliable results on all platforms and have enabled highly reproducible gene expression measurements. Thus, nearly all technical matters regarding microarray measurements are thought to have been resolved. Treatment stratification for molecular-targeted drugs can now be achieved based on the presence of somatic mutations, gene amplification, and/or protein overexpression. However, no clinically available biomarkers have been identified for molecular-targeted drugs using microarray analysis. Microarray data as a database for the gene expressions of clinical samples may be a critical issue, especially for the development of molecular-targeted treatments. In addition, microarray analysis during early-phase clinical trials for molecular-targeted drugs is considered to provide critical information, including proof-of-concept and confirmation of the inhibition of the target molecule. Meanwhile, OncotypeDX(®) and MammaPrint(®) assays have been developed to determine the benefits of chemotherapy for breast cancer patients. These multigene-based assays are commercially available and have shown encouraging results for treatment stratification or decision-making for treatment using cytotoxic drugs in clinical settings. During the development of these assays, numerous samples and efforts were required to create a model using multi-center or inter-group investigations. Based on the success of these models, the development of further assays for determining multigene expressions is likely to increase in the future. In the present article, we introduce our data on mutant epidermal growth factor receptor (EGFR) signaling and amplification of fibroblast growth factor receptor 2 (FGFR2) using microarray analysis, and treatment stratification and clinical applications using gene expression profiles for cancer treatments are discussed.

Topics: Antineoplastic Agents; Butadienes; Early Growth Response Protein 1; ErbB Receptors; Gene Expression Profiling; Genes, Neoplasm; HEK293 Cells; Humans; Microarray Analysis; Molecular Targeted Therapy; Mutation; Neoplasms; Nitriles; Protein Kinase Inhibitors; Quinazolines; Receptor, Fibroblast Growth Factor, Type 2; Signal Transduction; Tyrphostins

Refined cancer models are required if researchers are to assess the burgeoning number of potential targets for cancer therapeutics in a clinically relevant context that allows a fast turnaround. Here we use tumor-associated genetic pathways to transform primary human epithelial cells from the epidermis, oropharynx, esophagus and cervix into genetically defined tumors in a human three-dimensional (3D) tissue environment that incorporates cell-populated stroma and intact basement membrane. These engineered organotypic tissues recapitulated natural features of tumor progression, including epithelial invasion through basement membrane, a complex process that is necessary for biological malignancy in 90% of human cancers. Invasion was rapid and was potentiated by stromal cells. Oncogenic signals in 3D tissue, but not 2D culture, resembled gene expression profiles from spontaneous human cancers. We screened 3D organotypic neoplasia with well-characterized signaling pathway inhibitors to distill a clinically faithful cancer gene signature. Multitissue 3D human tissue cancer models may provide an efficient and relevant complement to current approaches to characterizing cancer progression.

Topics: Basement Membrane; Butadienes; Cell Transformation, Neoplastic; Chromones; Epithelial Cells; Gene Expression Profiling; Humans; Models, Biological; Morpholines; Neoplasm Invasiveness; Neoplasms; Nitriles; Stromal Cells; Tissue Engineering

The identification of self-renewing and multipotent neural stem cells (NSCs) in the mammalian brain holds promise for the treatment of neurological diseases and has yielded new insight into brain cancer. However, the complete repertoire of signaling pathways that governs the proliferation and self-renewal of NSCs, which we refer to as the 'ground state', remains largely uncharacterized. Although the candidate gene approach has uncovered vital pathways in NSC biology, so far only a few highly studied pathways have been investigated. Based on the intimate relationship between NSC self-renewal and neurosphere proliferation, we undertook a chemical genetic screen for inhibitors of neurosphere proliferation in order to probe the operational circuitry of the NSC. The screen recovered small molecules known to affect neurotransmission pathways previously thought to operate primarily in the mature central nervous system; these compounds also had potent inhibitory effects on cultures enriched for brain cancer stem cells. These results suggest that clinically approved neuromodulators may remodel the mature central nervous system and find application in the treatment of brain cancer.

Topics: Animals; Cell Survival; Cells, Cultured; Mice; Molecular Structure; Neoplasms; Neurons; Pharmaceutical Preparations; Sensitivity and Specificity; Stem Cells

Cell-cell contacts play important roles in the homeostasis of normal epithelium and in the steps of metastasis of tumor cells, although signaling mechanisms to regulate cell-cell contacts are unclear. In this study, we observed that phenotype of no cell-cell contacts in rat intestinal epithelial cell subline (RIE1-Sca) correlated with increased Erk1/2 signaling activity, compared to that of parental RIE1 cells growing in colonies. Furthermore, cell-cell contacts between RIE1-Sca cells were reformed by treatment with a specific MEK inhibitor (U0126), with translocation of ZO1 and beta-catenin to cell-cell contacts, without changes of their expression levels. U0126 treatment also increased EGFR phosphorylation in a ligand-independent manner. Pretreatment with EGFR kinase inhibitor abolished U0126 treatment-mediated EGFR phosphorylation, and expression of dominant negative H-Ras N17 allowed EGFR phosphorylation and cell-cell contacts even without U0126 treatment. Furthermore, the expression of a nonphosphorylatable EGFR Y5F mutant abolished U0126-mediated cell-cell contacts. U0126 treatment also caused less efficient wound healing by keeping monolayer integrity intact, compared to control untreated cells. This U0126-mediated reduction in wound healing was further altered either by pretreatment of EGFR kinase inhibitor or expression of H-Ras N17 or EGFR Y5F. Taken together, this study supports a unique mechanism of cell-cell contact formation through MEK/Erks inhibition-mediated EGFR phosphorylation.

Topics: Amino Acid Substitution; Animals; beta Catenin; Butadienes; Cell Communication; Cell Line, Tumor; Enzyme Inhibitors; ErbB Receptors; Genes, Dominant; Humans; MAP Kinase Signaling System; Membrane Proteins; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Mutation, Missense; Neoplasm Metastasis; Neoplasms; Nitriles; Phosphoproteins; Phosphorylation; Protein Processing, Post-Translational; Proto-Oncogene Proteins p21(ras); Rats; Wound Healing; Zonula Occludens-1 Protein

Vesicular stomatitis virus (VSV) can replicate in malignant cells more efficiently than in normal cells. Although the selective replication appears to be caused by defects in the interferon (IFN) system in malignant cells, the mechanisms which render these cells less responsive to IFN remain poorly understood. Here we present evidence that an activated RAS/Raf1/MEK/ERK pathway plays a critical role in the defects. NIH 3T3 or human primary cells stably expressing active RAS or Raf1 were rapidly killed by VSV. Although IFNalpha treatment no longer protected the RAS- or Raf1-overexpressing cells from VSV infection, responsiveness to IFNalpha was restored following treatment with the mitogen-activated protein kinase kinase (MEK) inhibitor U0126. Similarly, human cancer-derived cell lines became more responsive to IFNalpha in conjunction with U0126 treatment. Intriguingly, dual treatment with both IFNalpha and U0126 severely reduced the levels of viral RNAs in the infected cells. Moreover, cancer cells showed defects in inducing an IFNalpha-responsive factor, MxA, which is known to block VSV RNA synthesis, and U0126 restored the MxA expression. Our observations suggest that activation of the extracellular signal-regulated protein kinase (ERK) signaling leads to the defect in IFNalpha-mediated upregulation of MxA protein, which facilitates VSV oncolysis. In view of the fact that 30% of all cancers have constitutive activation of the RAS/Raf1/MEK/ERK pathway, VSV would be an ideal oncolytic virus for targeting such cancers.

Topics: Animals; Butadienes; Cell Line; Extracellular Signal-Regulated MAP Kinases; Humans; Interferon-alpha; MAP Kinase Signaling System; Mice; Mitogen-Activated Protein Kinase Kinases; Neoplasms; Nitriles; Protein Kinase Inhibitors; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins c-raf; ras Proteins; RNA Viruses; Vesicular stomatitis Indiana virus

Thalidomide has gained renewed interest as a cancer therapeutic due to its potential antiangiogenic effects. The thalidomide analogues CPS11 and CPS49 are active in preclinical angiogenesis assays and xenograft model systems, but the biochemical basis for these observations is unclear.. To address this question, we assessed the toxicity of these thalidomide analogues in cancer cells, endothelial cells, and genetically modified cells using assays that measure apoptotic and nonapoptotic cell death. Phosphospecific and native antibodies were used in immunoblotting and immunohistochemical experiments to assess the activation states of kinases that control cellular survival in vitro and in vivo.. CPS49 predominantly induced nonapoptotic cell death in lung cancer cells, prostate cancer cells, and endothelial cells in a dose-dependent manner, whereas CPS11 was not cytotoxic. CPS49 did not inhibit kinases that promote survival, such as Akt or extracellular signal-regulated kinase, but rather rapidly activated the stress kinase p38 pathway in both cancer cells and endothelial cells. CPS49 activated p38 in tumor xenografts. Using p38alpha-/- cells or an inhibitor of p38, we show that the presence and activation of p38alpha is important for cytotoxicity in all cell types examined.. Our studies identify a unifying mechanism of action for cytotoxicity of the tetraflourinated thalidomide analogue, CPS49, and suggest that activation of p38 could serve as a biomarker in clinical trials with CPS49.

Topics: Animals; Butadienes; Cell Death; Cell Line, Tumor; Cell Proliferation; Disease Models, Animal; Dose-Response Relationship, Drug; Drug Screening Assays, Antitumor; Endothelial Cells; Enzyme Inhibitors; Extracellular Signal-Regulated MAP Kinases; Humans; Imidazoles; Mice; Mice, SCID; Mitogen-Activated Protein Kinase 14; Neoplasms; Nitriles; Pyridines; Structure-Activity Relationship; Thalidomide; Xenograft Model Antitumor Assays

Expression of c-myc proto-oncogene is inappropriate in a wide range of human tumors, and is a downstream target of Ras/Raf/ERK pathway, which promotes c-Myc stability by enhancing c-Myc expression and activity. The aim of this study was to investigate whether the oncogenic phenotype in the human muscle-derived Rhabdomyosarcoma (RD) cell line and in non muscle-derived human tumor cell lines (SW403, IGR39 and PC3) can be blocked by disrupting the c-Myc pathway either by means of pharmacological MEK/ERK inhibition or by direct inactivation of the c-Myc protein.. We demonstrate that, in all the tumor cell lines used, the MEK/ERK inhibitor U0126 rapidly induces c-Myc de-phosphorylation, which is followed by a marked reduction in its expression level, by inhibition of proliferation and by reversion of anchorage-independent growth. These data suggest that the targeting of pathways controlling c-Myc expression or stability reverses deregulated growth of different tumor-derived cell lines. Indeed, in RD cells, we found a marked down-regulation of cyclins E2, A and B and CDK2, all of which are known to be targets of c-Myc. Moreover, ectopic MadMyc chimera, a c-Myc function antagonist, causes dramatic growth arrest, CDK and cyclin modulation as well as inhibition of anchorage-independent growth in RD cells, as occurs in U0126-treated cells. In particular, we found that the mere inhibition of c-Myc by MadMyc chimera rescues the myogenic program, MHC expression and the acquisition of the myogenic-like phenotype in RD cells.. Our data provide evidence of the key role played by the MEK/ERK pathway in the growth arrest and transformation phenotype of Rhabdomyosarcoma and of non muscle-derived tumor cell lines. In fact, MEK/ERK inhibitor, U0126, induces growth arrest, anchorage-dependent growth of these cell lines. In addition, the results of this study demonstrate that the direct inactivation of c-Myc by Mad/Myc chimera rescues myogenic program and leads to the reversal of the Rhabdomyosarcoma phenotype. In conclusion these data strongly suggest that the targeting of c-Myc by means of the MEK inhibitor can be tested as a promising strategy in anti-cancer therapy.

Topics: Butadienes; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Down-Regulation; Enzyme Inhibitors; Extracellular Signal-Regulated MAP Kinases; Flow Cytometry; Humans; Immunoblotting; Immunoprecipitation; Mitogen-Activated Protein Kinases; Muscle Neoplasms; Neoplasms; Nitriles; Phenotype; Plasmids; Proto-Oncogene Mas; Proto-Oncogene Proteins c-myc; Rhabdomyosarcoma, Embryonal; RNA Interference; Signal Transduction; Transfection

The transcription factor activator protein-1 (AP-1) has been implicated in a large variety of biological processes including oncogenic transformation. The tyrosine kinases of the epidermal growth factor receptor (EGFR) constitute the beginning of one signal transduction cascade leading to AP-1 activation and are known to control cell proliferation and differentiation. Drug discovery efforts targeting this receptor and other pathway components have centred on monoclonal antibodies and small molecule inhibitors. Resistance to such inhibitors has already been observed, guiding the prediction of their use in combination therapies with other targeted agents such as RNA interference (RNAi). This study examines the use of RNAi and kinase inhibitors for qualification of components involved in the EGFR/AP-1 pathway of ME180 cells, and their inhibitory effects when evaluated individually or in tandem against multiple components of this important disease-related pathway.. AP-1 activation was assessed using an ME180 cell line stably transfected with a beta-lactamase reporter gene under the control of AP-1 response element following epidermal growth factor (EGF) stimulation. Immunocytochemistry allowed for further quantification of small molecule inhibition on a cellular protein level. RNAi and RT-qPCR experiments were performed to assess the amount of knockdown on an mRNA level, and immunocytochemistry was used to reveal cellular protein levels for the targeted pathway components.. Increased potency of kinase inhibitors was shown by combining RNAi directed towards EGFR and small molecule inhibitors acting at proximal or distal points in the pathway. After cellular stimulation with EGF and analysis at the level of AP-1 activation using a beta-lactamase reporter gene, a 10-12 fold shift or 2.5-3 fold shift toward greater potency in the IC50 was observed for EGFR and MEK-1 inhibitors, respectively, in the presence of RNAi targeting EGFR.. EGFR pathway components were qualified as targets for inhibition of AP-1 activation using RNAi and small molecule inhibitors. The combination of these two targeted agents was shown to increase the efficacy of EGFR and MEK-1 kinase inhibitors, leading to possible implications for overcoming or preventing drug resistance, lowering effective drug doses, and providing new strategies for interrogating cellular signalling pathways.

Topics: Antibodies, Monoclonal; beta-Lactamases; Butadienes; Cell Differentiation; Cell Proliferation; Dose-Response Relationship, Drug; Enzyme Inhibitors; Epidermal Growth Factor; ErbB Receptors; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; Genes, Reporter; Humans; Immunohistochemistry; Inhibitory Concentration 50; MAP Kinase Kinase 1; Neoplasms; Nitriles; Oligonucleotides, Antisense; Phosphorylation; Quinazolines; Reverse Transcriptase Polymerase Chain Reaction; RNA Interference; RNA, Messenger; Signal Transduction; Transcription Factor AP-1; Transfection; Tyrphostins

Interactions between cancer cells and their microenvironment are critical for the development and progression of solid tumors. This study is the first to examine the role of all members of the ErbB tyrosine kinase receptors (epidermal growth factor receptor [EGFR], ErbB-2, ErbB-3, or ErbB-4), expressed singly or as paired receptor combinations, in the regulation of angiogenesis both in vitro and in vivo. Comparison of all receptor combinations reveals that EGFR/ErbB-2 and ErbB-2/ErbB-3 heterodimers are the most potent inducers of vascular endothelial growth factor (VEGF) mRNA expression compared with EGFR/ErbB-3, EGFR/ErbB-4, ErbB-2/ErbB-4, and ErbB-3/ErbB-4. Immunohistochemistry of tumor xenografts overexpressing these heterodimers shows increased VEGF expression and remarkably enhanced vascularity. Enhanced VEGF expression is associated with increased VEGF transcription. Deletional analysis reveals that ErbB-mediated transcriptional up-regulation of VEGF involves a hypoxia-inducible factor 1-independent responsive region located between nucleotides -88 to -66 of the VEGF promoter. Mutational analysis reveals that the Sp-1 and AP-2 transcription factor binding elements within this region are required for up-regulation of VEGF by heregulin beta1 and that this up-regulation is dependent on the activity of extracellular signal-related protein kinases. These results emphasize the biological implications of cell signaling diversity among members of the ErbB receptor family in regulation of the tumor microenvironment.

Topics: Adenocarcinoma; Animals; Butadienes; Cell Line; Dimerization; DNA-Binding Proteins; Endothelial Growth Factors; Enzyme Inhibitors; ErbB Receptors; Genes, Reporter; Humans; Immunohistochemistry; Intercellular Signaling Peptides and Proteins; Kruppel-Like Transcription Factors; Lymphokines; Mice; Mice, Nude; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinases; Neoplasm Transplantation; Neoplasms; Neovascularization, Pathologic; Neuregulin-1; Nitriles; Promoter Regions, Genetic; Receptor, ErbB-2; Receptor, ErbB-3; Receptor, ErbB-4; Recombinant Fusion Proteins; Signal Transduction; Sp1 Transcription Factor; Transcription Factors; Transcription, Genetic; Transplantation, Heterologous; Up-Regulation; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors

Constitutive activation of the ERK pathway is associated with the neoplastic phenotype of a relatively large number of human tumor cells. Blockade of the ERK pathway by treatment with PD98059, a specific inhibitor of mitogen-activated protein (MAP) kinase/ERK kinase (MEK), completely suppressed the growth of tumor cells in which the pathway is constitutively activated (RPMI-SE and HT1080 cells). Consistent with its prominent antiproliferative effect, PD98059 induced a remarkable G(1) cell cycle arrest, followed by a modest apoptotic response, in these tumor cells. Selective up-regulation of p27(Kip1) was observed after PD98059 treatment of RPMI-SE and HT1080 cells. Overexpression in RPMI-SE cells of either a kinase-negative form of MEK1 or wild-type MAP kinase phosphatase-3 also induced up-regulation of p27(Kip1). The up-regulation of p27(Kip1) correlated with increased association of p27(Kip1) with cyclin E-cyclin-dependent kinase (CDK) 2 complexes, a concomitant inhibition of cyclin E-CDK2 kinase activity, and a consequent decrease in the phosphorylation state of retinoblastoma protein, which would culminate in the marked G(1) cell cycle arrest observed in these tumor cells. These results suggest that the complete growth suppression that follows specific blockade of the ERK pathway in tumor cells in which the pathway is constitutively activated is mediated by up-regulation of p27(Kip1).

Topics: Antineoplastic Agents; Apoptosis; Butadienes; Enzyme Inhibitors; Flavonoids; G1 Phase; Humans; Mitogen-Activated Protein Kinases; Neoplasms; Nitriles; Signal Transduction; Tumor Cells, Cultured