u-0126 and Cell-Transformation--Neoplastic

The extracellular signal-regulated kinase (ERK) signaling pathway is essential for cell proliferation and is frequently deregulated in human tumors such as melanoma. Melanoma remains incurable despite the use of conventional chemotherapy; consequently, development of new therapeutic agents for melanoma is highly desirable. Here, we carried out a chemical genetic screen using a fission yeast phenotypic assay and showed that ACA-28, a synthetic derivative of 1'-acetoxychavicol acetate (ACA), which is a natural ginger compound, effectively inhibited the growth of melanoma cancer cells wherein ERK MAPK signaling is hyperactivated due to mutations in the upstream activating regulators. ACA-28 more potently inhibited the growth of melanoma cells than did the parental compound ACA. Importantly, the growth of normal human epidermal melanocytes (NHEM) was less affected by ACA-28 at the same 50% inhibitory concentration. In addition, ACA-28 specifically induced apoptosis in NIH/3T3 cells which were oncogenically transformed with human epidermal growth factor receptor-2 (HER2/ErbB2), but not in the parental cells. Notably, the ACA-28-induced apoptosis in melanoma and HER2-transformed cells was abrogated when ERK activation was blocked with a specific MEK inhibitor U0126. Consistently, ACA-28 more strongly stimulated ERK phosphorylation in melanoma cells, as compared in NHEM. ACA-28 might serve as a promising seed compound for melanoma treatment.

Topics: 3T3 Cells; Animals; Antineoplastic Agents; Apoptosis; Benzyl Alcohols; Butadienes; Cell Line, Tumor; Cell Transformation, Neoplastic; Enzyme Inhibitors; Extracellular Signal-Regulated MAP Kinases; Gene Expression Regulation, Enzymologic; Humans; MAP Kinase Kinase 1; Melanocytes; Melanoma; Mice; Nitriles; Phosphorylation; Signal Transduction

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

Tungsten carbide-cobalt (WC-Co) nanoparticle composites have wide applications because of their hardness and toughness. WC-Co was classified as "probably carcinogenic" to humans by the International Agency for Research on Cancer (IARC) in 2003. It is believed that the toxicity and carcinogenesis of WC-Co is associated with particle size. Recent studies demonstrated that the tumor suppressor gene programmed cell death 4 (PDCD4) and its upstream regulator miR-21 have been considered as oncogenes for novel cancer prevention or anticancer therapies. The present study examined the effects of WC-Co nanoparticles on miR-21-PDCD4 signaling in a mouse epidermal cell line (JB6 P+). The results showed that (i) exposure of JB6 cells to WC-Co stimulated a increase of miR-21 generation; (ii) WC-Co also caused inhibition of PDCD4, a tumor suppressor protein and downstream target of miR-21, expression in JB6 cells; (iii) inhibition of ERKs with ERK inhibitor U0126 significantly reversed WC-Cominus;induced PDCD4 inhibition, but inhibition of p38 with p38 inhibitor SB203580 did not; and (iv) ROS scavengers, N-acetyl-L-cysteine and catalase, blocked the inhibitory effect of WC-Co on PDCD4 expression, while superoxide dismutase promoted the inhibitory effect. These findings demonstrate that WC-Co nanoparticles induce miR-21 generation, but inhibit PDCD4 production, which may be mediated through ROS, especially endogenous H2O2, and ERK pathways. Unraveling the complex mechanisms associated with these events may provide insights into the initiation and progression of WC-Co-induced carcinogenesis.

Topics: Animals; Apoptosis Regulatory Proteins; Butadienes; Cell Line; Cell Transformation, Neoplastic; Cobalt; Disease Models, Animal; Enzyme Inhibitors; Epidermis; Hydrogen Peroxide; Imidazoles; In Vitro Techniques; MAP Kinase Signaling System; Mice; MicroRNAs; Mitogen-Activated Protein Kinase Kinases; Nanoparticles; Nitriles; Occupational Exposure; Pyridines; Reactive Oxygen Species; RNA-Binding Proteins; Signal Transduction; Tungsten Compounds

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

The runt-domain transcription factor Runx3 plays crucial roles during development such as regulating gene expression. It has been shown that Runx3 is involved in neurogenesis, thymopoiesis and functions like a tumor suppressor. Runx3 null mouse die soon after birth as a result of multiple organ defects. Runx3 null mouse lung shows an abnormal phenotype and loss of Runx3 induced remodeling in the lung. Interestingly, lung adenocarcinoma is observed in Runx3 heterozygous mice at 18 months of age. During lung development various cellular and molecular events occur such as cell proliferation, cell death, differentiation and epithelial-mesenchymal transition (EMT). To understand the specific lethal events in Runx3 null mice, we examined cellular and molecular networks involved in EMT, and EMT inducers were quantified by RT-qPCR during lung development. Excessive EMT was observed in lungs at PN1 day in Runx3 null mice and PN18 months in Runx3 heterozygous mice. Pharmacologic inhibition of EMT was used to curb tumor progression. In this study, U0126 was injected to pregnant mouse for inhibition of pERK signaling. After U0126 treatment, life spans of newborn mice were increased and lung hyperplasia was partially rescued by down-regulated cell proliferation and EMT. Our data suggest that Runx3 is involved in crucial regulation of alveolar differentiation and tumor suppression in developing mouse lung.

Topics: Adenocarcinoma; Adenocarcinoma of Lung; Animals; Butadienes; Cell Differentiation; Cell Transformation, Neoplastic; Core Binding Factor Alpha 3 Subunit; eIF-2 Kinase; Epithelial-Mesenchymal Transition; Female; Gene Expression Regulation, Developmental; Lung; Lung Neoplasms; Mice; Mice, Knockout; Nitriles; Pregnancy; Pulmonary Alveoli; Signal Transduction

Lapatinib, a dual HER2 and EGFR tyrosine kinase inhibitor is highly active in HER2+ breast cancer. However, its efficacy is limited by either primary or acquired resistance. Although mutations in ras genes are rarely found in breast cancer, H-ras overexpression is frequently observed. Moreover, genetic alterations that do not directly involve ras such as Brk amplification, ultimately result in increased ras signaling. Using SKBR3 cells, a HER2+ breast cancer cell line that is naturally devoid of mutations in PI3KCA, PTEN, BRAF, and ras we show that both H-ras overexpression and expression of an oncogenic ras allele (ras V12) reduce susceptibility to lapatinib in analogy to what observed with activating PI3KCA mutations and with a constitutively active form of Akt. Importantly, we found that resistance to lapatinib due to ras overexpression or to ras V12 is overcome by MEK inhibition with U0126, suggesting a key role for the MEK-Erk pathway in ras-induced resistance. Similar results were obtained in BT474 cells, another HER+ breast cancer cell line. Therefore, our data indicate that overexpressed/mutated ras may act as a biological modifier of the response to lapatinib. Combining MEK inhibitors with lapatinib may help overcome this form of resistance and increase the efficacy of lapatinib in these tumors.

Topics: Antineoplastic Agents; Breast Neoplasms; Butadienes; Cell Line, Tumor; Cell Survival; Cell Transformation, Neoplastic; Drug Resistance, Neoplasm; Enzyme Inhibitors; Female; Genes, ras; Humans; Immunoblotting; Lapatinib; Mitogen-Activated Protein Kinase Kinases; Mutation; Nitriles; Quinazolines; Receptor, ErbB-2; Signal Transduction

The resorcylic acid lactone hypothemycin has been shown to inactivate protein kinases by binding to a cysteine conserved in 46 protein kinases, including mitogen-activated protein kinase kinase (MEK), extracellular signal-regulated kinase (ERK) and platelet-derived growth factor receptor (PDGFR). We assessed the selectivity of hypothemycin in cellular contexts. Hypothemycin normalized the morphology and inhibited anchorage-independent growth of Ki-ras transformed normal rat kidney (NRK) cells with selectivity and potency comparable to or greater than that of the MEK inhibitor U0126. In Ki-ras-transformed and phorbol 12-myristate 13-acetate (PMA)-treated NRK cells, hypothemycin blocked ERK activation but showed a minimal effect on autophosphorylation of protein kinase D1 (PKD1), another kinase containing the conserved cysteine. Hypothemycin potently inhibited PDGFR autophosphorylation and activation of the MEK-ERK pathway in platelet-derived growth factor (PDGF)-treated NRK cells. However, the phosphoinositide-3-kinase (PI3K) pathway was only modestly attenuated. Hypothemycin also inhibited growth factor- and anchorage-independent growth of human cancer cell lines with a constitutively active MEK-ERK pathway. Although hypothemycin has the potential to inactivate various protein kinases, the results indicate that in intracellular environments, hypothemycin can inhibit the MEK-ERK axis with sufficient selectivity to normalize transformed phenotypes of cells dependent on this pathway.

Topics: Animals; Butadienes; Cell Line, Tumor; Cell Transformation, Neoplastic; Extracellular Signal-Regulated MAP Kinases; Fibroblasts; Flavonoids; Growth Inhibitors; Humans; Kidney; MAP Kinase Signaling System; Mitogen-Activated Protein Kinase Kinases; Nitriles; Protein Kinase Inhibitors; Rats; Zearalenone

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

Many cancers, including breast cancer, harbor loss-of-function mutations in the catalytic domain of phosphatase and tensin homologue deleted on chromosome 10 (PTEN) or have reduced PTEN expression through loss of heterozygosity and/or epigenetic silencing mechanisms. However, specific phenotypic effects of PTEN inactivation in human cancer cells remain poorly defined without a direct causal connection between the loss of PTEN function and the development or progression of cancer. To evaluate the biological and clinical relevance of reduced or deleted PTEN expression, a novel in vitro model system was generated using human somatic cell knockout technologies. Targeted homologous recombination allowed for a single and double allelic deletion, which resulted in reduced and deleted PTEN expression, respectively. We determined that heterozygous loss of PTEN in the nontumorigenic human mammary epithelial cell line MCF-10A was sufficient for activation of the phosphoinositide 3-kinase/AKT and mitogen-activated protein kinase pathways, whereas the homozygous absence of PTEN expression led to a further increased activation of both pathways. The deletion of PTEN was able to confer growth factor-independent proliferation, which was confirmed by the resistance of the PTEN(-/-) MCF-10A cells to small-molecule inhibitors of the epidermal growth factor receptor. However, neither heterozygous nor homozygous loss of PTEN expression was sufficient to promote anchorage-independent growth, but the loss of PTEN did confer apoptotic resistance to cell rounding and matrix detachment. Finally, MCF-10A cells with the reduction or loss of PTEN showed increased susceptibility to the chemotherapeutic drug doxorubicin but not paclitaxel.

Topics: Anoikis; Antibiotics, Antineoplastic; Antineoplastic Agents, Phytogenic; Apoptosis; Blotting, Western; Butadienes; Cell Proliferation; Cell Transformation, Neoplastic; Chromones; Doxorubicin; Enzyme Inhibitors; Epithelial Cells; Female; Flow Cytometry; Humans; Integrases; Mammary Glands, Human; Mitogen-Activated Protein Kinases; Morpholines; Nitriles; Paclitaxel; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; PTEN Phosphohydrolase; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Signal Transduction

Elevated expression and aberrant activation of the src oncogene are strongly associated with cancer initiation and progression, thereby making Src a promising molecular target for anti-cancer therapy. Through drug screening using a temperature-inducible v-Src-transformed epithelial cell line, we found that andrographolide could suppress v-Src-induced transformation and down-regulate v-Src protein expression. In addition, actin cable dissolution and E-cadherin down-regulation, features of transformed phenotype, are perturbed by andrographolide. Moreover, andrographolide promoted v-Src degradation via a ubiquitin-dependent manner. Although andrographolide treatment altered the tyrosine phosphorylation pattern in v-Src-expressing cells, it did not directly affect the kinase activity of v-Src. Both the Erk and phosphatidylinositol 3-kinase signaling pathways were strongly inhibited in andrographolide-treated v-Src cells. However, only MKK inhibitors (PD98059 and U0126) were able to cause a non-transformed morphology similar to that of andrographolide-treated v-Src cells. Moreover, overexpression of constitutively active MKK1 in v-Src cells blocked andrographolide-mediated morphological inhibition. Interestingly, andrographolide treatment could also reduce the protein level of the c-Src truncation mutant (Src531), an Src mutant originally identified from human colon cancer cells. In summary, we demonstrated that andrographolide antagonized v-Src action through promotion of v-Src protein degradation. Furthermore, attenuation of the Erk1/2 signaling pathway is essential for andrographolide-mediated inhibition of v-Src transformation. Our results demonstrate that andrographolide can act as a v-Src inhibitor and reveal a novel action mechanism of andrographolide.

Topics: Antiviral Agents; Butadienes; Cell Line, Tumor; Cell Transformation, Neoplastic; Colonic Neoplasms; Diterpenes; Enzyme Inhibitors; Epithelial Cells; Flavonoids; Gene Expression Regulation, Neoplastic; Hot Temperature; Humans; MAP Kinase Signaling System; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Mutation; Nitriles; Oncogene Protein pp60(v-src); Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Phosphorylation; Tyrosine; Ubiquitin; Ubiquitination

Bone morphogenetic proteins (BMPs) are multifunctional cytokines that regulate key developmental processes, but are also overexpressed in many carcinomas. To assess whether BMPs would influence the three-dimensional architecture of epithelial structures, we took advantage of an in vitro model in which mammary epithelial cells form alveolar-like spherical cysts in collagen gels. We found that BMP-4 has a dramatic, biphasic effect on the organization of epithelial cysts. When added in the concentration range of 1-10 ng/ml, the cytokine abrogates lumen formation and induces the outgrowth of multiple invasive cord-like structures. At higher concentrations (20-100 ng/ml), BMP-4 additionally disrupts cell-cell adhesion, resulting in cyst disintegration and scattering of individual cells into the surrounding collagen matrix. The finding that BMP-4 subverts the ability of mammary epithelial cells to form polarized lumen-containing structures and endows them with invasive properties supports the involvement of this cytokine in the progression of breast cancer.

Topics: Animals; Anthracenes; Bone Morphogenetic Protein 4; Bone Morphogenetic Proteins; Butadienes; Cell Transformation, Neoplastic; Cells, Cultured; Epithelial Cells; Female; Humans; Imidazoles; Mammary Glands, Animal; Mice; Mitogen-Activated Protein Kinases; Neoplasm Invasiveness; Nitriles

Retroviral integration mutagenesis and treatment with the frameshift mutagen ICR191 were used to transform v-H-ras expressing PB-3c cells to interleukin-3 (IL-3) independence. Six clones displayed viral integrations into the 3' region of the IL-3 gene thus acting post-transcriptionally by disrupting the AU-rich instability element. Two clones contained reverse orientation integration into the raf-1 gene revealing an enhancer insertion mechanism. Growth by this mechanism was sensitive to the Raf-1 inhibitor BAY 43-9006 and the Mek inhibitor U0126. Following treatment with ICR191, IL-3-independent clones were recovered and studied by cell fusion. With 21/22 clones, IL-3 independence resulted from a recessive mechanism as cellular hybrids with parental cells reverted to IL-3 dependence. Recessive clone D2c displayed increased phospho-Erk1/2 levels and was growth sensitive to U0126, but not to BAY43-9006. The single dominant clone, D5a, showed no signs of mitogen-activated protein kinases pathway activation but displayed constitutive phosphorylation of Stat5. We conclude that PB-3c has several options to acquire IL-3 growth autonomy involving transcriptional or post-transcriptional mechanisms affecting the distal regulators Erk or Stat5. The reported panel of independent dominant and recessive transformants should provide a useful tool for inhibitor profiling.

Topics: Aminacrine; Benzenesulfonates; Butadienes; Cell Transformation, Neoplastic; Clone Cells; Frameshift Mutation; Genes, Dominant; Genes, Recessive; Hematopoiesis; Humans; Interleukin-3; MAP Kinase Kinase Kinases; Mutagenesis, Insertional; Neoplastic Stem Cells; Niacinamide; Nitriles; Nitrogen Mustard Compounds; Phenylurea Compounds; Proto-Oncogene Proteins c-raf; Pyridines; Retroviridae; Sorafenib; STAT5 Transcription Factor; Transfection

Protein kinase C (PKC) zeta has been implicated as a mediator of epidermal growth factor (EGF) receptor (EGFR) signaling in certain cell types. Because EGFR is ubiquitously expressed in squamous cell carcinomas of the head and neck (SCCHN) and plays a key role in tumor progression, we determined whether PKCzeta is required for tumor cell proliferation and viability. Examination of total and phosphorylated PKCzeta expression in normal oral mucosa, dysplasia, and carcinoma as well as SCCHN tumor cell lines revealed a significant increase in activated PKCzeta expression from normal to malignant tissue. PKCzeta activity is required for EGF-induced extracellular signal-regulated kinase (ERK) activation in both normal human adult epidermal keratinocytes and five of seven SCCHN cell lines. SCCHN cells express constitutively activated EGFR family receptors, and inhibition of either EGFR or mitogen-activated protein kinase (MAPK) activity suppressed DNA synthesis. Consistent with this observation, inhibition of PKCzeta using either kinase-dead PKCzeta mutant or peptide inhibitor suppressed autocrine and EGF-induced DNA synthesis. Finally, PKCzeta inhibition enhanced the effects of both MAPK/ERK kinase (U0126) and broad spectrum PKC inhibitor (chelerythrine chloride) and decreased cell proliferation in SCCHN cell lines. The results indicate that (a) PKCzeta is associated with SCCHN progression, (b) PKCzeta mediates EGF-stimulated MAPK activation in keratinocytes and SCCHN cell lines, (c) PKCzeta mediates EGFR and MAPK-dependent proliferation in SCCHN cell lines; and (d) PKCzeta inhibitors function additively with other inhibitors that target similar or complementary signaling pathways.

Topics: Alkaloids; Amino Acid Sequence; Benzophenanthridines; Butadienes; Carcinoma, Squamous Cell; Cell Growth Processes; Cell Line, Tumor; Cell Transformation, Neoplastic; DNA, Neoplasm; Enzyme Activation; Epidermal Growth Factor; ErbB Receptors; Head and Neck Neoplasms; Humans; Keratinocytes; Mitogen-Activated Protein Kinases; Molecular Sequence Data; Mouth Mucosa; Mouth Neoplasms; Nitriles; Phenanthridines; Protein Kinase C; Protein Kinase Inhibitors

We report for the first time that over-expression of Myc suppresses mitogen-activated ERK kinase (MEK)/extracellular regulated kinase (ERK) signalling in chick embryo fibroblasts (CEF). Myc does not interfere with individual components of the signalling cascade, since efficient signal propagation via MEK and ERK in Myc-infected CEF can be seen. However, using the Myc-binding domain (MBD) of Bin-1, which binds to and negatively regulates the activity of Myc, we selectively suppressed Myc-induced apoptosis, without affecting its transforming properties. This was accompanied by a restoration in MEK/ERK signalling, suggesting a critical role for this pathway in regulating apoptosis in these cells. This was also confirmed using a specific pharmacological inhibitor of MEK. Experiments with conditioned media suggest that over-expression of Myc may inhibit autocrine growth factor production, which can be restored by co-expression of MBD. Although the identity of the growth factor(s) is not known, we propose a feedback mechanism whereby Myc interferes with growth factor signalling.

Topics: Adaptor Proteins, Signal Transducing; Animals; Apoptosis; Autocrine Communication; Butadienes; Cell Nucleus; Cell Transformation, Neoplastic; Cells, Cultured; Chick Embryo; Down-Regulation; Extracellular Signal-Regulated MAP Kinases; Growth Substances; MAP Kinase Signaling System; Mice; Mitogen-Activated Protein Kinase Kinases; Nerve Tissue Proteins; Nitriles; Protein Kinase Inhibitors; Protein Structure, Tertiary; Proto-Oncogene Proteins c-myc; Tumor Suppressor Proteins

In the United States, oral cancer accounts for more deaths annually than cervical cancer, leukemias, or Hodgkin's lymphoma. Studies have shown that aberrations of chromosome 18q develop with tumor progression and are associated with significantly decreased survival in head and neck cancer patients. The G-protein-coupled receptor, galanin receptor 1 (GALR1), maps to this region of chromosome 18q. Although the role of GALR1 has been well characterized in neuronal cells, little is known regarding this receptor in non-neuronal cells. In this study, the expression, mitogenic function, and signaling mechanism of GALR1 are investigated in normal and malignant oral epithelial cells. mRNA expression was determined via reverse transcriptase-PCR. Protein quantification was done via immunoblot analysis and enzyme-linked immunosorbent assay. For functional and signaling studies, an inhibitory antibody was generated to the N-terminal ligand binding domain of GALR1. GALR1 protein and mRNA expression and GAL secretion were detected at variable levels in immortalized human oral keratinocytes and human oropharyngeal squamous cell carcinoma cell lines. Upon competitive inhibition of GALR1, proliferation was up-regulated in immortalized and malignant keratinocytes. Furthermore, studies with the inhibitory antibody and U0126, the MAPK inhibitor, show that GALR1 inhibits proliferation in immortalized and malignant keratinocytes by inactivating the MAPK pathway. GALR1s inhibitory effects on proliferation in epithelial cells raises the possibility that inactivation or disregulation of this receptor can lead to uncontrolled proliferation and neoplastic transformation.

Topics: Animals; Antibodies; Antineoplastic Agents; Binding, Competitive; Butadienes; Carcinoma, Squamous Cell; Cell Proliferation; Cell Transformation, Neoplastic; Cells, Cultured; CHO Cells; Chromosomes, Human, Pair 18; Cricetinae; Culture Media, Conditioned; Enzyme Activation; Enzyme Inhibitors; Enzyme-Linked Immunosorbent Assay; Galanin; Humans; Immunoblotting; Keratinocytes; Ligands; MAP Kinase Signaling System; Models, Biological; Mouth Neoplasms; Nitriles; Protein Binding; Protein Structure, Tertiary; Receptor, Galanin, Type 1; Receptor, Galanin, Type 2; Receptor, Galanin, Type 3; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Signal Transduction; Time Factors; Transfection; Up-Regulation

The apoptosis-promoting protein Par-4 has been shown to be down-regulated in Ras-transformed NIH 3T3 fibroblasts through the Raf/MEK/ERK MAPK pathway. Because mutations of the ras gene are most often found in tumors of epithelial origin, we explored the signaling pathways utilized by oncogenic Ras to down-regulate Par-4 in RIE-1 and rat ovarian surface epithelial (ROSE) cells. We determined that constitutive activation of the Raf, phosphatidylinositol 3-kinase, or Ral guanine nucleotide exchange factor effector pathway alone was not sufficient to down-regulate Par-4 in RIE-1 or ROSE cells. However, treatment of Ras-transformed RIE-1 or ROSE cells with the MEK inhibitors U0126 and PD98059 increased Par-4 protein expression. Thus, although oncogenic Ras utilizes the Raf/MEK/ERK pathway to down-regulate Par-4 in both fibroblasts and epithelial cells, Ras activation of an additional signaling pathway(s) is required to achieve the same outcome in epithelial cells. Methylation-specific PCR showed that the par-4 promoter is methylated in Ras-transformed cells through a MEK-dependent pathway and that treatment with the DNA methyltransferase inhibitor azadeoxycytidine restored Par-4 mRNA transcript and protein levels, suggesting that the mechanism for Ras-mediated down-regulation of Par-4 is by promoter methylation. Support for this possibility is provided by our observation that Ras transformation was associated with up-regulation of Dnmt1 and Dnmt3 DNA methyltransferase expression. Finally, ectopic Par-4 expression significantly reduced Ras-mediated growth in soft agar, but not morphological transformation, highlighting the importance of Par-4 down-regulation in specific aspects of Ras-mediated transformation of epithelial cells.

Topics: Agar; Alleles; Animals; Apoptosis; Apoptosis Regulatory Proteins; Azacitidine; Blotting, Northern; Blotting, Western; Butadienes; Cell Line; Cell Line, Tumor; Cell Transformation, Neoplastic; Decitabine; DNA (Cytosine-5-)-Methyltransferase 1; DNA (Cytosine-5-)-Methyltransferases; DNA Methylation; DNA Methyltransferase 3A; DNA, Complementary; Down-Regulation; Enzyme Inhibitors; Epithelial Cells; Female; Fibroblasts; Flavonoids; Genetic Vectors; Humans; Intracellular Signaling Peptides and Proteins; Mice; Mutation; NIH 3T3 Cells; Nitriles; Ovary; Phosphatidylinositol 3-Kinases; Polymerase Chain Reaction; raf Kinases; Rats; RNA; RNA, Messenger; Signal Transduction; Up-Regulation

Anthocyanins are the chemical components that give the intense color to many fruits and vegetables, such as blueberries, red cabbages and purple sweet potatoes. Extensive studies have indicated that anthocyanins have strong antioxidant activities. To investigate the mechanism of anthocyanidins as an anticancer food source, six kinds of anthocyanidins representing the aglycons of most anthocyanins, were used to examine their effects on tumor promotion in mouse JB6 cells, a validated model for screening cancer chemopreventive agents and elucidating the molecular mechanisms. Of the six anthocyanins tested, only those with an ortho-dihydroxyphenyl structure on the B-ring suppressed 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced cell transformation and activator protein-1 transactivation, suggesting that the ortho-dihydroxyphenyl may contribute to the inhibitory action. Delphinidin, but not peonidin, blocked the phosphorylation of protein kinases in the extracellular signal-regulated protein kinase (ERK) pathway at early times and the c-Jun N-terminal kinase (JNK) signaling pathway at later times. p38 kinase was not inhibited by delphinidin. Furthermore, two mitogen-activated protein kinase (MAPK) specific inhibitors (SP600125 for JNK and UO126 for ERK) could specifically block the activation of JNK and ERK and cell transformation. Those results demonstrate that anthocyanidins contribute to the inhibition of tumorigenesis by blocking activation of the MAPK pathway. These findings provide the first molecular basis for the anticarcinogenic action of anthocyanidins.

Topics: Animals; Anthocyanins; Anthracenes; Anticarcinogenic Agents; Butadienes; Cell Transformation, Neoplastic; MAP Kinase Signaling System; Mice; Mitogen-Activated Protein Kinases; Nitriles; Phosphorylation; Structure-Activity Relationship; Superoxide Dismutase; Tetradecanoylphorbol Acetate; Transcription Factor AP-1

Human hepatocellular carcinoma (HCC) is associated with increased expression and activity of mitogen-activated protein kinase (MAPK) signaling intermediates (ie, MEK, ERK).. We determined the effects of MEK-ERK signaling on proliferation, cell cycle, apoptosis, and tumorigenicity of HCC in vitro. HCC cell lines were treated with MEK enzyme-specific inhibitors, PD098059 and U0126, and ERK1,2 oligonucleotide antisense.. In the HCC cells examined, MEK inhibitors blocked ERK1,2 phosphorylation without a change in total ERK expression. ERK1,2 oligonucleotide antisense inhibited ERK1,2 protein expression. PD098059, U0126, and ERK1,2 oligonucleotide antisense each inhibited HCC cellular proliferation in a concentration-dependent manner. Cell cycle, apoptosis, and tumorigenicity were examined in Hep3B and HepG2 cell lines. MEK enzyme inhibition resulted in anticancer effects through cell cycle arrest, increased apoptosis, and decreased tumorigenicity in these cell lines. U0126 exhibited more potent inhibition of ERK1,2 phosphorylation and had more pronounced anticancer effects in both cell lines. Correspondingly, HepG2 cells, the cell line more sensitive to ERK1,2 phosphorylation inhibition, sustained more pronounced anticancer effects with treatment. But Hep3B cells were more sensitive to ERK1,2 antisense-mediated decreases in ERK1,2 protein expression and correspondingly, their growth was inhibited to a greater degree than the HepG2 cells. MEK enzyme inhibition had downstream effects on the expression of the antiapoptotic protein survivin in both cell lines.. These data suggest that there are multiple anticancer effects of blocking MEK-ERK signaling, and that these depend on both the susceptibility of the cells and the ability of the treatment to effect a selective block of MEK-ERK signaling in HCC cells.

Topics: Apoptosis; Blotting, Western; Butadienes; Carcinoma, Hepatocellular; Cell Count; Cell Division; Cell Transformation, Neoplastic; Dose-Response Relationship, Drug; Enzyme Inhibitors; Flavonoids; Humans; Liver Neoplasms; MAP Kinase Kinase Kinase 1; MAP Kinase Kinase Kinases; Mitogen-Activated Protein Kinases; Nitriles; Oligonucleotides, Antisense; Phosphorylation; Signal Transduction; Tumor Cells, Cultured; Tumor Stem Cell Assay

Activation of the transcription factor AP-1 (activator protein-1) is required for tumor promotion and maintenance of malignant phenotype. A number of AP-1-regulated genes that play a role in tumor progression have been identified. However, AP-1-regulated genes driving tumor induction are yet to be defined. Previous studies have established that expression of a dominant-negative c-Jun (TAM67) inhibits phorbol 12-tetradecanoyl-13-acetate (TPA)-induced AP-1 transactivation as well as transformation in mouse epidermal JB6/P+ cells and tumor promotion in mouse skin carcinogenesis. In this study, we utilized the tumor promotion-sensitive JB6/P+ cells to identify AP-1-regulated TAM67 target genes and to establish causal significance in transformation for one target gene. A 2700 cDNA microarray was queried with RNA from TPA-treated P+ cells with or without TAM67 expression. Under conditions in which TAM expression inhibited TPA-induced transformation, microarray analysis identified a subset of six genes induced by TPA and suppressed by TAM67. One of the identified genes, the high-mobility group protein A1 (Hmga1) is induced by TPA in P+, but not in transformation-resistant P cells. We show that TPA induction of the architectural transcription factor HMGA1 is inhibited by TAM67, is extracellular-signal-regulated kinase (ERK)-activation dependent, and is mediated by AP-1. HMGA1 antisense construct transfected into P+ cells blocked HMGA1 protein expression and inhibited TPA-induced transformation indicating that HMGA1 is required for transformation. HMGA1 is not however sufficient as HMGA1a or HMGA1b overexpression did not confer transformation sensitivity on P- cells. Although HMGA1 expression is ERK dependent, it is not the only ERK-dependent event required for transformation because it does not suffice to rescue ERK-deficient P- cells. Our study shows (a) TAM 67 when it inhibits AP-1 and transformation, targets a relatively small number of genes; (b) HMGA1, a TAM67 target gene, is causally related to transformation and therefore a potentially important target for cancer prevention.

Topics: Animals; Butadienes; Cell Line; Cell Transformation, Neoplastic; Clone Cells; Cyclin D1; Disease Susceptibility; DNA, Complementary; Epidermal Cells; Epidermis; Gene Expression Profiling; Genes, jun; HMGA1a Protein; HMGA1b Protein; MAP Kinase Kinase Kinase 1; MAP Kinase Kinase Kinases; MAP Kinase Signaling System; Mice; Mice, Transgenic; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinases; Nitriles; Oligonucleotide Array Sequence Analysis; Oligonucleotides, Antisense; Osteopontin; Proto-Oncogene Proteins c-jun; Sialoglycoproteins; Tetradecanoylphorbol Acetate; Transcription Factor AP-1; Transcription, Genetic

Although Vav can act as a guanine nucleotide exchange factor for RhoA, Rac1, and Cdc42, its transforming activity has been ascribed primarily to its ability to activate Rac1. However, because activated Vav, but not Rac-specific guanine nucleotide exchange factors, exhibits very potent focus-forming transforming activity when assayed in NIH 3T3 cells, Vav transforming activity must also involve activation of Rac-independent pathways. In this study, we determined the involvement of other Rho family proteins and their signaling pathways in Vav transformation. We found that RhoA, Rac1, and Cdc42 functions are all required for Vav transforming activity. Furthermore, we determined that Vav activation of nuclear factor-kappaB and the Jun NH2-terminal kinase mitogen-activated protein kinase (MAPK) is necessary for full transformation by Vav, whereas p38 MAPK does not seem to play an important role. We also determined that Vav is a weak activator of Elk-1 via a Ras- and MAPK/extracellular signal-regulated kinase kinase-dependent pathway, and this activity was essential for Vav transformation. Thus, we conclude that full Vav transforming activation is mediated by the activation of multiple small GTPases and their subsequent activation of signaling pathways that regulate changes in gene expression. Because Vav is activated by the epidermal growth factor receptor and other tyrosine kinases involved in cancer development, defining the role of aberrant Vav signaling may identify activities of receptor tyrosine kinases important for human oncogenesis.

Topics: Animals; Blotting, Western; Butadienes; cdc42 GTP-Binding Protein; Cell Culture Techniques; Cell Cycle Proteins; Cell Proliferation; Cell Transformation, Neoplastic; DNA-Binding Proteins; Enzyme Inhibitors; ets-Domain Protein Elk-1; Gene Expression Regulation, Neoplastic; Genes, Dominant; Genes, Reporter; Genetic Vectors; GTP Phosphohydrolases; MAP Kinase Signaling System; Mice; NF-kappa B; NIH 3T3 Cells; Nitriles; p38 Mitogen-Activated Protein Kinases; Phosphorylation; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-vav; rac1 GTP-Binding Protein; ras Proteins; rhoA GTP-Binding Protein; Signal Transduction; Transcription Factors; Transfection

U0126, a recently introduced mitogen-activated protein kinase [corrected] (MAPK)/extracellular signal-regulated kinase kinase inhibitor reversed morphology and inhibited anchorage-independent growth of Ki-ras-transformed rat fibroblasts. Immunoblot analyses with phosphospecific antibodies indicated that in addition to MAPK, U0126 suppressed activation of p70(S6K), but not Akt, at concentrations at which it normalized the transformed phenotypes. Another MAPK/extracellular signal-regulated kinase kinase inhibitor, PD98059, showed only marginal effects on p70S6K phosphorylation and did not effectively block Ki-ras-induced transformation. However, simultaneous inhibition of the MAPK pathway and the p70S6K pathway by PD98059 in conjunction with the p70S6K inhibitor rapamycin essentially restored the normal phenotype. U0126 or the combination of PD98059 and rapamycin flattened morphology of v-src-transformed cells, but did not reverse anchorage independence, although activation of both MAPK and p706K was blocked. The results suggest that normalization of Ki-ras-induced transformed phenotypes by U0126 is a consequence of concurrent inhibition of the MAPK and p70S6K pathways. Intervention of other pathway(s) appears to be required to completely antagonize transformation by v-src. Simultaneous blockade of more than one signal transduction pathway by combining selective inhibitors might be effective in suppressing uncontrolled tumorigenic growth.

Topics: Animals; Butadienes; Cell Line, Transformed; Cell Size; Cell Transformation, Neoplastic; Contact Inhibition; Drug Synergism; Enzyme Activation; Fibroblasts; Flavonoids; MAP Kinase Signaling System; Mitogen-Activated Protein Kinase Kinases; Mitogen-Activated Protein Kinases; Nitriles; Oncogene Protein p21(ras); Phenotype; Phosphorylation; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; Rats; Ribosomal Protein S6 Kinases; Sirolimus

Overexpression of ErbB-2/Neu has been causally associated with mammary epithelial transformation. Here we report that blockade of the epidermal growth factor receptor (EGFR) kinase with AG-1478 markedly delays breast tumor formation in mouse mammary tumor virus (MMTV)/Neu + MMTV/transforming growth factor alpha bigenic mice. This delay was associated with inhibition of EGFR and Neu signaling, reduction of cyclin-dependent kinase 2 (Cdk2) and mitogen-activated protein kinase (MAPK) activities and cyclin D1, and an increase in the levels of the Cdk inhibitor p27(Kip1). In addition, BrdUrd incorporation into tumor cell nuclei was prevented with no signs of tumor cell apoptosis. These observations prompted us to investigate the stability of p27. Recombinant p27 was degraded rapidly in vitro by untreated but not by AG-1478-treated tumor lysates. Proteasome depletion of the tumor lysates, addition of the specific MEK1/2 inhibitor U-0126, or a T187A mutation in recombinant p27 all prevented p27 degradation. Cdk2 and MAPK precipitates from untreated tumor lysates phosphorylated recombinant wild-type p27 but not the T187A mutant in vitro. Cdk2 and MAPK precipitates from AG-1478-treated tumors were unable to phosphorylate p27 in vitro. These data suggest that increased signaling by ErbB receptors up-regulates MAPK activity, which, in turn, phosphorylates and destabilizes p27, thus contributing to dysregulated cell cycle progression.

Topics: Animals; Butadienes; CDC2-CDC28 Kinases; Cell Cycle Proteins; Cell Division; Cell Transformation, Neoplastic; Cyclin D1; Cyclin-Dependent Kinase 2; Cyclin-Dependent Kinase Inhibitor p27; Cyclin-Dependent Kinases; Cysteine Endopeptidases; Dimerization; DNA; Down-Regulation; ErbB Receptors; Female; Humans; Mammary Neoplasms, Experimental; Mammary Tumor Virus, Mouse; Mice; Mice, Transgenic; Microtubule-Associated Proteins; Mitogen-Activated Protein Kinase Kinases; Mitogen-Activated Protein Kinases; Multienzyme Complexes; Nitriles; Phosphorylation; Proteasome Endopeptidase Complex; Protein Serine-Threonine Kinases; Quinazolines; Receptor, ErbB-2; Signal Transduction; Time Factors; Transforming Growth Factor alpha; Tumor Cells, Cultured; Tumor Suppressor Proteins; Tyrphostins

The chimeric gene EWS/FLI-1, the hallmark of the Ewing's sarcoma and primitive neuroectodermal tumor family, encodes a fusion protein with enhanced transcriptional activation properties and preserved recognition of canonical ETS binding sites. Although EWS/FLI-1 alters the expression of various genes, the precise mechanism by which EWS/FLI-1 acts as an oncogene remains to be defined. In this study we report that members of the mitogen-activated protein kinase (MAPK) signaling pathway, ERK1 and ERK2, are constitutively activated in NIH 3T3 cells expressing EWS/FLI-1. Interference with ERK activation by either highly specific inhibitors of MEK1 or a dominant negative ras mutant profoundly impaired the ability of EWS/FLI-1 to transform NIH3T3 cells to growth in semi-solid medium. An EWS/FLI-1 mutant defective in DNA-binding and transcriptional activation failed to activate ERK and was also defective in 3T3 cell transformation. Constitutive ERK activation was also evident in several human Ewing's sarcoma tumor-derived cell lines. Interestingly, cells expressing the type II EWS/FLI-1 fusion, recently demonstrated more potent in transcriptional activation, showed even greater MAPK activation than cells expressing the more common type I fusion. These results implicate ERK activation in EWS/FLI-1 transformation and suggest that this signaling pathway may be important in the pathogenesis of Ewing's sarcoma. Oncogene (2000) 19, 4523 - 4530.

Topics: 3T3 Cells; Animals; Binding Sites; Bone Neoplasms; Butadienes; Cell Transformation, Neoplastic; Colony-Forming Units Assay; DNA; Enzyme Activation; Enzyme Inhibitors; Flavonoids; Genes, ras; Humans; Imidazoles; MAP Kinase Kinase 1; Mice; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinase Kinases; Mitogen-Activated Protein Kinases; Mutation; Nitriles; Oncogene Proteins, Fusion; Protein Serine-Threonine Kinases; Proto-Oncogene Protein c-fli-1; Pyridines; RNA-Binding Protein EWS; Sarcoma, Ewing; Transcription Factors; Tumor Cells, Cultured