Oncogenic tyrosine kinase NPM/ALK induces activation of the rapamycin-sensitive mTOR signaling pathway

The mechanisms of cell transformation mediated by the nucleophosmin (NPM)/anaplastic lymphoma kinase (ALK) tyrosine kinase are only partially understood. Here, we report that cell lines and native tissues derived from the NPM/ALK-expressing T-cell lymphoma display persistent activation of mammalian target of rapamycin (mTOR) as determined by phosphorylation of mTOR targets S6rp and 4E-binding protein 1 (4E-BP1). The mTOR activation is serum growth factor-independent but nutrient-dependent. It is also dependent on the expression and enzymatic activity of NPM/ALK as demonstrated by cell transfection with wild-type and functionally deficient NPM/ALK, small interfering RNA (siRNA)-mediated NPM/ALK depletion and kinase activity suppression using the inhibitor WHI-P154. The NPM/ALK-induced mTOR activation is transduced through the mitogen-induced extracellular kinase (MEK)/extracellular signal-regulated kinase (ERK) signaling pathway and, to a much lesser degree, through the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) pathway. Accordingly, whereas the low-dose PI3K inhibitor wortmannin and Akt inhibitor III profoundly inhibited Akt phosphorylation, they had a very modest effect on S6rp and 4E-BP1 phosphorylation. In turn, MEK inhibitors U0126 and PD98059 and siRNA-mediated depletion of either ERK1 or ERK2 inhibited S6rp phosphorylation much more effectively. Finally, the mTOR inhibitor rapamycin markedly decreased proliferation and increased the apoptotic rate of ALK+TCL cells. These findings identify mTOR as a novel key target of NPM/ALK and suggest that mTOR inhibitors may prove effective in therapy of ALK-induced malignancies.     

Prolonged extracellular signal-regulated kinase 1/2 activation during fibroblast growth factor 1- or heregulin beta1-induced antiestrogen-resistant growth of breast cancer cells is resistant to mitogen-activated protein/extracellular regulated kinase kinase inhibitors

Increased growth factor receptor signaling is implicated in antiestrogen-resistant breast tumors suggesting that abrogation of such signaling could restore or prolong sensitivity to antihormonal agents. Activation of the mitogen-activated protein/extracellular regulated kinase kinase (MEK)-extracellular regulated kinase (ERK)1/2 cascade is a common component of such pathways. We investigated the ability of the MEK activation inhibitor U0126 to block the increased growth of estrogen receptor-positive MCF-7 breast cancer cells caused by fibroblast growth factor 1 (FGF-1), heregulin beta1 (HRGbeta1), and epidermal growth factor (EGF) in the presence of the pure antiestrogen ICI 182780 (Faslodex; fulvestrant). We found that either FGF-1 or HRGbeta1 but not EGF substantially reduced the inhibitory effects of U0126 on growth and ERK1/2 activation, including the combined inhibitory effects of U0126 and ICI 182780. FGF-1 and HRGbeta1 also reduced the inhibition of ERK1/2 phosphorylation by the MEK inhibitors PD98059 and PD184161. Interestingly, a transiently transfected dominant-negative MEK1 completely abrogated activation of a coexpressed green fluorescent protein-ERK2 reporter by all three of the factors. Despite a short-lived activation of Ras and Raf-1 by all three of the growth factors, both FGF-1 and HRGbeta1, unlike EGF, induced a prolonged activation of MEK and ERK1/2 in these cells. Thus, activation of FGF-1- and HRGbeta1-specific signaling causes MEK-dependent prolonged activation of ERK1/2, which is incompletely susceptible to known MEK inhibitors. We also demonstrate that the cytosolic phospholipase A2 inhibitor arachidonyl trifluoro methyl ketone and the pan PKC inhibitor bisindolymaleimide abrogated U0126-resistant phosphorylation of ERK1/2 induced by HRGbeta1 but not by FGF-1. Phosphorylation of ERK5 by all three of the factors was also resistant to U0126 suggesting that its activation is not sufficient to overturn growth inhibition due to diminished ERK1/2 activation. Therefore, therapy combining antiestrogens and MEK inhibitors may be ineffective in some antiestrogen-resistant estrogen receptor-positive breast cancers.     

Relationship between the expression of extracellular signal-regulated kinase 1/2 and the dissociation of pancreatic cancer cells: Involvement of ERK1/2 in the dissociation status of cancer cells

Mitogen-activated protein kinase kinase 2 (MEK2), the upstream kinase of extracellular signal-regulated kinase 1/2 (ERK1/2) was previously isolated as cancer cell dissociation related factor. In this study, to further clarify the regulatory mechanism of cancer cell dissociation, two hamster (PC-1.0 and PC-1) and human (Capan-2 and AsPC-1) pancreatic cancer cell lines were analyzed immunocytochemically with anti-ERK1, anti-ERK2 and anti-phosphorylated ERK1/2 (p-ERK1/2) antibodies. U0126 (a MEK1/2 inhibitor) significantly suppressed ERK2 and p-ERK1/2 expressions in PC-1.0 and AsPC-1 cells (P<0.05). Cancer cell dissociation factor (DF) markedly induced ERK2 and p-ERK1/2 expressions in PC-1 and Capan-2 cells (P<0.05), and the induced ERK2 and p-ERK1/2 expressions were inhibited by subsequent U0126-treatment (P<0.05). Simultaneously, light microscopic images showed that DF clearly induced cell dissociation in PC-1 and Capan-2 cells, while U0126-treatment induced cell aggregation in these pancreatic cancer cells. ERK2 activation is closely involved in cell dissociation of pancreatic cancer cells.     

Significant anti-proliferation of human endometrial cancer cells by combined treatment with a selective COX-2 inhibitor NS398 and specific MEK inhibitor U0126

The extracellular signal-regulated kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) pathway plays a critical role in the anticancer action in vitro. ERK1/2 activation or phosphorylation is responsible for increased cyclooxygenase-2 (COX-2) protein expression in some cancer cells treated with selective COX-2 inhibitor NS398. We determined the effect of NS398 on ERK signaling and the synergistic effect of combined treatment with NS398 and a specific MEK inhibitor U0126 on three human endometrial cancer cell lines: Ishikawa, HEC-1A and AN3CA cells. Results showed that NS398 and U0126 individually, and especially the combination of both exhibited profound anti-proliferation of all three cell lines in a time- and concentration-dependent manner by [3-(4, 5)-dimethylthiazol-z-yl]-2, 5-diphenyl tetrazolium bromide (MTT) assay. The phosphorylated ERK1/2 was up-regulated in HEC-1A and AN3CA cells, but the COX-2 protein expression was unchanged in the three cancer cell lines treated with NS398 alone. However, both phosphorylated ERK1/2 and COX-2 protein expression were concentration-dependently decreased in all three cell types by combined treatment with NS398 and U0126 assessed by western blot analysis. Simultaneously, the combination of NS398 and U0126 resulted in 2-fold increase in apoptosis of all three lines over that by the individual alone, and enhanced G0/G1 phase arrest of Ishikawa and HEC-1A cells induced by U0126 treatment determined by flow cytometry. The synergistic and complementary effects of combining NS398 and U0126 were found to be associated with activation of caspase-3, alterations of Bcl-2 family proteins and cell cycle regulatory proteins detected by western blot analysis. Taken together, these findings correlate with blocking MEK-ERK signaling cascade and down-regulating COX-2 protein expression in endometrial cancer cells with combination treatment of NS398 and U0126, suggesting that the combinatory use of NS398 and specific MEK inhibitors may be valuable for chemotherapy or chemoprevention of human endometrial cancer.     

Inhibition of extracellular-signal regulated kinases 1/2 is required for apoptosis of human colon cancer cells in vitro by sulindac metabolites

Nonsteroidal anti-inflammatory drugs (NSAIDs) including sulindac have shown potent chemopreventive and tumor regressive effects against colorectal cancer, the second leading cause of cancer death in the United States. However, the mechanisms by which sulindac inhibits tumor cell growth are not completely understood. We previously reported that sulindac metabolites inhibit the mitogen-activated protein/extracellular signal-regulated kinase kinase/extracellular signal-regulated kinase (MEK/ERK) signaling cascade in colorectal cancer cell lines at doses that induce apoptosis, and inhibition of MEK/ERK activity with U0126 is sufficient to induce apoptotic cell death. To determine whether inhibition of MEK/ERK activity is necessary for sulindac-induced apoptosis of human colon cancer cells, stable transfectants were created that express an activated MEK1 gene in HT29 cells. HT29-MEK1(R4F) clones displayed a 10- to 20-fold increase in MEK1 activity compared with control HT29-pCEP4 clones. When compared with control HT29-pCEP4 clones, HT29-MEK1(R4F) clones were resistant to both apoptosis and inhibition of ERK1/2 phosphorylation induced by sulindac metabolites. These results suggest that inhibition of MEK/ERK signaling is necessary for the induction of apoptosis by sulindac metabolites.     

Importance of MEK-1/-2 signaling in monocytic and granulocytic differentiation of myeloid cell lines.

Activation of the MEK/ERK/MAP kinase signaling pathway promotes the proliferation and survival of hematopoietic cells. The kinases MEK-1, MEK-2, ERK-1/MAPK and ERK-2/MAPK are activated by phosphorylation at specific sites, and these events can be monitored using phospho-specific antibodies. In this report we examined the importance of the MEK/ERK/MAP kinase pathway in the monocytic and granulocytic differentiation of myeloid cell lines. Induction of monocytic differentiation in HL-60 cells by treatment with phorbol 12-myristate 13-acetate (PMA) led to rapid and sustained activation of MEK-1/-2, ERK-1/MAPK and ERK-2/MAPK, while induction of granulocytic differentiation by retinoic acid (RA) caused similar activation of MEK-1/-2 and ERK-2/MAPK, but not ERK-1/MAPK. The total levels of these kinases were not affected during the course of differentiation along either pathway. Pretreatment of cells with 5 microM of the MEK-1/-2-specific inhibitor U0126 abrogated PMA- or RA-induced activation of ERK-1/MAPK and ERK-2/MAPK. Importantly, pretreatment of HL-60 cells with U0126 was found to potently inhibit both monocytic and granulocytic differentiation, as assessed by cytochemical staining for non-specific esterase or nitroblue tetrazolium reduction, flow cytometric analysis of myeloid surface markers, and immunoblotting for the cell cycle inhibitor p21 WAF1/Cip1. Similar results were seen in U937 cells, where U0126 inhibited PMA-induced monocytic differentiation, and in 32D cells, where G-CSF-induced granulocytic differentiation was inhibited by U0126 pretreatment. Additional experiments revealed that inhibition of MEK-1/-2 in HL-60 cells resulted in nearly complete inhibition of differentiation-induced cell death during monocytic differentiation. By contrast, U0126 only partially inhibited cell death resulting from granulocytic differentiation. Taken together, our findings demonstrate that the MEK/ERK/MAP kinase signaling pathway is activated, and plays a critical role, during both monocytic and granulocytic differentiation of myeloid cell lines.     

Involvement of ERK1/2 and p38 MAP kinase in doxorubicin-induced uPA expression in human RC-K8 lymphoma and NCI-H69 small cell lung carcinoma cells

We previously demonstrated the doxorubicin-induced urokinase-type plasminogen activator (uPA) expression in human RC-K8 lymphoma cells and NCI-H69 small cell lung carcinoma cells in which reactive oxygen species might be involved. Western blotting analysis revealed phosphorylation/activation of mitogen-activated protein (MAP) kinases, such as extracellular signal-regulated kinase (ERK) 1/2, p38 MAP kinase and stress-activated protein kinase/c-jun N-terminal protein kinase (SAPK/JNK) in doxorubicin-treated RC-K8 and H69 cells, and, therefore, we attempted to identify the MAP kinases implicated in doxorubicin-induced uPA expression by the use of their specific inhibitors. U0126, SB202190 and JNKI-1, inhibitors for MAPK kinase, (MEK) 1/2, p38 MAP kinase and SAPK/JNK, respectively, specifically and clearly inhibited their corresponding kinases. U0126 and SB202190, but not JNKI-1, almost completely inhibited the doxorubicin-induced uPA expression in both RC-K8 and H69 cells. However, U0126 rather enhanced the doxorubicin-induced activation of caspase-3 and poly ADP-ribose polymerase (PARP), and U0126 itself activated caspase-3 and PARP. Interestingly, JNKI-1 inhibited the doxorubicin-induced activation of caspase-3 and PARP. Therefore, doxorubicin treatment activates the above three kinases, but different MAP kinase signaling is responsible in the doxorubicin-induced caspase activation and expression of uPA. Thus, we could possibly manipulate the direction of doxorubicin-induced MAP kinase activation and the effects of doxorubicin on the tumor cell biology by the use of MAP kinase inhibitors.     

ERK/MAPK activation involves hypoxia‐induced MGr1‐Ag/37LRP expression and contributes to apoptosis resistance in gastric cancer

We previously demonstrated that hypoxia increased the hypoxia‐inducible factor (HIF‐1)–dependent MGr1‐Ag/37LRP expression, which enhanced adhesion of gastric cancer cells to laminin, inhibited drug‐induced apoptosis and caused cell adhesion–mediated drug resistance (CAM‐DR). Here, we investigated the role of extracellular‐regulated kinase (ERK) 1/2 in the signaling mechanisms underlying these events. We found that hypoxia activated ERK activity in vitro and in vivo. Overexpression of mitogen‐activated protein kinase (MAPK) kinase (MEK), which preferentially activated ERK, mimics, in a nonadditive way, hypoxia‐induced activity of MGr1‐Ag/37LRP promoter and expression of MGr1‐Ag/37LRP. Furthermore, U0126, the MEK inhibitor, inhibited hypoxia‐ and MEK‐induced MGr1‐Ag/37LRP promoter activity in a dose‐dependent manner. MEK inhibition also reversed hypoxia‐ and MEK‐induced HIF‐1 protein and its activity in a dose‐dependent manner. We also investigated reactive oxygen species signaling this response. Exogenous addition of H₂O₂ was sufficient to activate ERK in a dose‐dependent profile. Reactive oxygen species scavengers of H₂O₂ significantly inhibited hypoxia‐induced ERK or HIF‐1 activation and sequential expression of MGr1‐Ag/37LRP. We also investigated the signaling in hypoxia‐induced cell adhesion and apoptosis induced by vincristine. Hypoxia significantly enhanced adhesion of SGC7901 cells to laminin in a time‐dependent manner, which might be inhibited by the MEK inhibitor U0126 and MGr1‐Ag/37LRP siRNA. Consistent with results of adhesion assay, hypoxia‐resistant apoptosis might be reversed by U0126 in a dose‐dependent manner. Our results suggest that hypoxia‐elicited MGr1‐Ag/37LRP expression activated by HIF‐1 depends on ERK activation. These events are dependent of reactive oxygen intermediates.     

The MEK/ERK pathway mediates COX-2-selective NSAID-induced apoptosis and induced COX-2 protein expression in colorectal carcinoma cells

Nonsteroidal antiinflammatory drugs (NSAIDs) can prevent colorectal tumorigenesis in humans and in rodents. In vitro and in vivo studies indicate that one of their principal antineoplastic avenues is the induction of apoptosis. We have shown previously that NS-398, which selectively inhibits cyclooxygenase-2 (COX-2) over cyclooxygenase-1, induces apoptosis of colorectal tumour cells and elevates COX-2 protein expression. Here, we have determined that the extracellular signal-regulated kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) pathway mediates these effects of NS-398. Treatment of HT29 colorectal carcinoma cells with 75 microM NS-398 caused activation of ERK-1/-2 but not of the p38 and c-Jun N-terminal kinase (JNK) mitogen-activated protein kinases. This was apparent at 24 hr and maintained at 72 hr. U0126, a specific inhibitor of the ERK-activating kinases MEK-1/-2, prevented the activation of ERK induced by NS-398 and blocked the increase in COX-2 protein expression seen when HT29 cells were treated with NS-398 alone. The activation of ERK by NS-398 preceded and accompanied a decrease in attached cell yield and an increase in apoptosis. U0126 dose-dependently protected HT29 cells from these antiproliferative effects of NS-398, indicating an antiproliferative role for sustained ERK-1/-2 activation in response to this NSAID. These results point to a key role for the MEK/ERK signalling pathway in mediating the effects of a COX-2-selective NSAID on colorectal carcinoma cells.     

Analysis of invasion-metastasis mechanism in pancreatic cancer: involvement of tight junction transmembrane protein occludin and MEK/ERK signal transduction pathway in cancer cell dissociation

Mitogen-activated protein kinase kinase 2 (MEK2) was detected as an invasion-metastasis related factor between highly invasive (PC-1.0) and weakly invasive (PC-1) pancreatic cancer cell lines in our previous study. On the other hand, tight junction (TJ) was found to be correlated with carcino-genesis and tumor development. In this study, the expressions and correlation of TJ transmembrane protein occludin and MEK/extracellular signal-regulated kinase (ERK) signaling pathway were analyzed to clarify the regulatory mechanism of cell dissociation in pancreatic cancer cells. Two hamster (PC-1.0 and PC-1) and human (AsPC-1 and CAPAN-2) pancreatic cancer cell lines were analyzed immunocytochemically with anti-occludin, phosphorylated MEK1/2 (p-MEK1/2), phosphorylated ERK1/2 (p-ERK1/2) antibodies. MEK1/2 inhibitor U0126 significantly induced the expression of occludin at the cell-cell junction and substantially suppressed the p-MEK1/2 and p-ERK1/2 expressions in PC-1.0 and AsPC-1 cells. In contrast, dissociation factor (DF) treatment obviously disrupted the occludin expressions at the sites of cell-cell junction and markedly induced the p-MEK1/2 and p-ERK1/2 expressions in PC-1 and CAPAN-2 cells. In addition, occludin expressions at cell-cell junction were restored and p-MEK1/2 and p-ERK1/2 expressions were suppressed by subsequent U0126-treatment in DF treated PC-1 and CAPAN-2 cells. Correspondingly, light microscopic images showed that DF induced the dissociation of cell island-like colonies in PC-1 and CAPAN-2 cells, and U0126-treatment induced cell aggregation in these pancreatic cancer cells. Occludin is involved in the cell dissociation in pancreatic cancer cells. Moreover, MEK/ERK signaling pathway probably regulates the cell dissociation status of pancreatic cancer through influencing the intracellular localization and expression of occludin.     

Potentiation of the antitumor effects of both selective cyclooxygenase-1 and cyclooxygenase-2 inhibitors in human hepatic cancer cells by inhibition of the MEK/ERK pathway

The molecular mechanisms behind the anti-neoplastic effects of non-steroidal anti-inflammatory drugs (NSAIDs) are not completely understood and cannot be explained by the inhibition of the cyclooxygenase (COX) enzymes COX-1 and COX-2 alone. We previously reported that both the selective COX-1 inhibitor SC-560 and the selective COX-2 inhibitor CAY10404 exhibit anti-tumor effects in human hepatoma cells. NSAID inhibitors have many COX-independent actions and, among others, the mitogen-activated protein kinase (MAPK) pathways are targets for NSAIDs. Here, we examined the role of MEK/ERK1/2 signaling in the anti-neoplastic effects of both selective COX-1 and COX-2 inhibitors in two human hepatoma cell lines. Treatment of hepatoma cells with the selective COX-1 inhibitor SC-560, as well as with the selective COX-2 inhibitor CAY10404, was associated with activation of ERK1/2 in a time- and dose-dependent manner. Treatment with COX-1 and COX-2 inhibitors in the presence of the selective MEK1/2 inhibitor U0126 effectively suppressed ERK1/2 activation and combinations of either SC-560 or CAY10404 with U0126 resulted in synergistic effects on cell growth inhibition and induction of apoptosis. In HuH-6 hepatoma cells the combination-induced apoptosis was associated with caspase-9 and -3 activation, PARP cleavage, release of cytochrome c from the mitochondria into the cytosol and down-regulation of survivin and beta-catenin levels. In conclusion, our study showed that growth inhibitory concentrations of selective COX-1 and COX-2 inhibitors increased ERK1/2 phosphorylation in hepatoma cells, and that inhibition of the MEK/ERK signaling pathway potentiates the antitumor activity of both types of inhibitors. Therefore, our results provide preclinical support for a combined chemotherapeutic approach with selective NSAIDs and MEK inhibitors for the treatment of hepatocellular carcinoma.     

U0126, a mitogen-activated protein kinase kinase inhibitor, inhibits the invasion of human A375 melanoma cells

The anti-invasive ability of the mitogen-activated protein kinase (MAPK) kinase inhibitor, U0126, was examined in human A375 melanoma cells in vitro. The effect was compared to that of PD98059, another commonly used MEK (MAPK kinase) inhibitor. U0126 or PD98059 showed a dose-dependent inhibition of A375 cell invasion through growth factor-reduced Matrigel. U0126 was more potent than PD98059 in suppressing tumor cell invasion. Both compounds significantly decreased urokinase plasminogen activator (uPA) and matrix metalloproteinases-9 (MMP-9) concentrations in conditioned media. At 5 microM, U0126 inhibited phosphorylation of the MEK 1/2 to a non-detectable level within 24 h. The phosphorylation of extracellular signal-related kinase 1/2 was also dramatically suppressed by the treatment with 10 microM U0126 or 40 microM PD98059. Both compounds suppressed the protein expression of c-Jun, but not c-Fos. The expression of uPA and MMP-9 was also inhibited. Our data suggest that U0126 is an effective agent in inhibiting human A375 melanoma cell invasion and that the effect is partially due to the decreased production of uPA and MMP-9.     

Apoptosis of liver cancer cells by vitamin K2 and enhancement by MEK inhibition

Vitamin K2 (VK2) is an anti-proliferative agent toward a variety of cancer including hepatocellular carcinoma (HCC). Because the growth inhibitory effect of VK2 to HCC has not been established yet, we investigated it in HCC cells in vitro. VK2 inhibited growth of Hep3B, but not of HepG2, HLF, and Huh6. VK2 induced the cell cycle arrest at the G1 phase and involvement of apoptosis was suggested because the sub-G1 fraction appeared in flow cytometric analysis and nuclear condensation and fragmentation appeared after VK2 treatment. VK2 activated extracellular signal-regulated kinase (ERK)1/2 in a mitogen-activated ERK-regulating kinase (MEK)-dependent manner in Hep3B and Huh6, but not in HepG2 and HLF. When ERK1/2 was inhibited by U0126, apoptosis by VK2 in Hep3B, but not in Huh6, was significantly enhanced. However, Western blot analysis revealed that neither apoptosis induction by VK2 nor enhancement of apoptosis by U0126 was mediated by caspase activation. These data demonstrated that VK2 induced apoptosis and activated the MEK/ERK1/2 signaling pathway in a cell-type specific manner, and a MEK inhibitor could augment the cell death in these cells.     

NO donor and MEK inhibitor synergistically inhibit proliferation and invasion of cancer cells

Nitric oxide (NO) shows tumoricidal activity. We had previously reported that NO downregulates the phosphatidylinositol-3-kinase/Akt pathway, but upregulates the MEK/ERK pathway downstream of growth factor signaling. We hypothesized that NO donor and MEK inhibitor in combination synergistically inhibit the viability of cancer cells compared to either NO donor or MEK inhibitor alone. We determined the effects of S-nitrosoglutathione (GSNO, NO-donor) and U0126 (MEK inhibitor) on insulin-like growth factor-I (IGF-I) and epidermal growth factor (EGF) signaling, proliferation and invasion in cancer cell lines. GSNO inhibits phosphorylation of IGF-I receptor (IGF-IR), EGF receptor (EGFR) and Akt, but upregulates ERK1/2 phosphorylation in MIAPaCa-2 and HCT-116 cells after stimulation by IGF-I and EGF. On the other hand, U0126 inhibits phosphorylation of ERK1/2, but upregulates phosphorylation of IGF-IR and EGFR in MIAPaCa-2 and HCT-116 cells. The combination of GSNO and U0126 downregulates phosphorylation of IGF-IR, EGFR, Akt and ERK1/2 after stimulation by IGF-I and EGF. GSNO as well as U0126, inhibits the proliferation of MIAPaCa-2, HCT-116, Panc-1, MCF-7, HT-29 and AGS cells in a dose-dependent manner. GSNO and U0126 in combination synergistically inhibit proliferation and invasion of cancer cells. These results indicate that the combined treatment of NO donor and MEK inhibitor may be promising in cancer therapy.     

Pharmacological targeting of the constitutively activated MEK/MAPK-dependent signaling pathway in glioma cells inhibits cell proliferation and migration

Activated mitogen-activated protein kinase MAPK cascade leading to ERK1/2 phosphorylation is expressed in the majority of glial neoplasms and negatively correlates with survival time of patients. Here we show that ERK1/2 kinases are constitutively activated in glioma cell lines and stem cell-enriched primary cultures of glioblastoma. Pharmacological targeting of the activated MEK/ERK1/2 module with the MEK inhibitor U0126 attenuates cell cycle progression (11 out of 11 cell lines), impairs single (7 out of 10) and collective cell migration (9 out of 11) and abolishes single cell emigration from monolayers (4 out of 9). Attacking the activated MEK/ERK1/2 module thus partially blocks the tumorigenic potential of glial cancer cells on different levels and strongly suggests the application of combination molecularly targeted therapies to interfere more efficiently with glial tumor development and progression.     

Stimulation of cultured colon 26 cells with TNF-alpha promotes lung metastasis through the extracellular signal-regulated kinase pathway

We investigated the influence of TNF-alpha on the metastasis of cancer cells. Treatment of cultured colon 26 cells with TNF-alpha enhanced metastatic properties including production of MMP-9, adhesion, migration and invasion. Cells treated with TNF-alpha in vitro showed marked potential to metastasize to the lung and liver in vivo. U0126, an inhibitor of MEK1/2, inhibited the TNF-alpha-induced activation of extracellular signal-regulated kinase 1/2 (ERK1/2) and the metastatic properties in vitro without affecting cell proliferation. In addition, pretreatment with U0126 in vitro completely abrogated the increased lung metastasis of TNF-alpha-treated cells. These results indicate that TNF-alpha-induced activation of cancer cells through the ERK pathway is sufficient for the enhanced metastatic potential of colon 26 cells.     

The p38 pathway inhibitor SB202190 activates MEK/MAPK to stimulate the growth of leukemia cells

In this study, the biological effects of signal transduction inhibitors on leukemia cells were examined. We found that the p38 inhibitor SB202190 enhanced the growth of THP-1 and MV4-11 cells. To determine the pathway affected by SB202190, we examined the 50% effective dose (ED(50)) values for THP-1 cell growth in combination with several inhibitors. In the presence of SB202190, the ED(50) values for the farnesyltransferase inhibitor FPT inhibitor II and MEK inhibitor U0126 were significantly decreased. Western blot analysis revealed that SB202190 increased the phosphorylation of C-Raf and extracellular regulated kinase (ERK), suggesting that Ras-Raf-MEK-mitogen-activated protein kinase (MAPK) pathway activation is involved in the leukemia cell growth induced by SB202190.     

Activation of mammalian target of rapamycin in transformed B lymphocytes is nutrient dependent but independent of Akt, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase, insulin growth factor-I, and serum

The study examines the preponderance and mechanism of mammalian target of rapamycin (mTOR) activation in three distinct types of transformed B lymphocytes that differ in expression of the EBV genome. All three types [EBV-immortalized cells that express a broad spectrum of the virus-encoded genes (type III latency; EBV+/III), EBV-positive cells that express only a subset of the EBV-encoded genes (EBV+/I), and EBV-negative, germinal center-derived cells (EBV-)] universally displayed activation of the mTOR signaling pathway. However, only the EBV+/III transformed B cells displayed also activation of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway that is considered to be the key activator of mTOR and of the mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK) kinase (MEK)/ERK pathway that coactivates one of the immediate targets of mTOR, p70 S6K1. Activation of the PI3K/Akt and MEK/ERK, but not of the mTOR pathway, was inhibited by serum withdrawal and restored by insulin growth factor-I. In contrast, activation of mTOR, but not PI3K/Akt and MEK/ERK, was sensitive to nutrient depletion. Both direct Akt (Akt inhibitors I-III) and a PI3K inhibitor (wortmannin at 1 nmol/L) suppressed Akt phosphorylation without significantly affecting mTOR activation. Furthermore, rapamycin, a potent and specific mTOR inhibitor, suppressed profoundly proliferation of cells from all three types of transformed B cells. U0126, a MEK inhibitor, had a moderate antiproliferative effect only on the EBV+/III cells. These results indicate that mTOR kinase activation is mediated in the transformed B cells by the mechanism(s) independent of the PI3K/Akt signaling pathway. They also suggest that inhibition of mTOR signaling might be effective in therapy of the large spectrum of B-cell lymphomas.     

Pro-apoptotic role of the MEK/ERK pathway in ursodeoxycholic acid-induced apoptosis in SNU601 gastric cancer cells

Ursodeoxycholic acid (UDCA) has been regarded as a suppressor of gastrointestinal cancer, but the mechanisms underlying its antitumor effects are not fully understood. Previously, we reported the antitumor effect of UDCA by demonstrating that UDCA induces apoptosis of gastric cancer cells. Bile acids are known to activate the ERK pathway and ERK is a representative oncogenic kinase in cancer cells. Here, we investigated the role of ERK in UDCA-induced gastric cancer cell apoptosis. We found that UDCA enhanced the phosphorylation of ERK1/2 and MEK1/2. The prevention of MEK by the pharmacologic inhibitors PD98059 and U0126, resulted in decreased UDCA-induced apoptosis as shown by the reduction of apoptotic body formation, caspase-8 activity, and caspase-3, -6 and PARP cleavage, indicating that ERK exerts pro-apoptotic activity upon exposure to UDCA. In addition, U0126 reduced UDCA-triggered TNF-related apoptosis-inducing ligand receptor 2 (TRAIL-R2/DR5) expression. In gene silencing studies, we observed that RNA interference of ERK2 decreased apoptosis and reduced DR5 overexpression. Lipid raft disrupting agent, methyl-β-cyclodextrin, blunted the phosphorylation of ERK1/2, indicating that ERK activation is regulated in a lipid raft-dependent manner. On the other hand, tumor-promoting bile acid, deoxycholic acid (DCA), also phosphorylated ERK in SNU601 cells. However, the DCA-triggered ERK pathway exerted anti-apoptotic function in the cells. Suppression of the ERK pathway enhanced DCA-induced apoptosis, and ERK activation was observed to be lipid raft-independently controlled. These results indicated that UDCA and DCA may cause differential responses in gastric cancer cells through the ERK signaling molecule. Thus, ERK activation may be a possible mechanism by which UDCA and DCA represent differential activities in gastrointestinal cancer.