Identification of an EGFRvIII-JNK2-HGF/c-Met-Signaling Axis Required for Intercellular Crosstalk and Glioblastoma Multiforme Cell Invasion.

Glioblastoma multiforme (GBM) is the most aggressive and common form of adult brain cancer. Current therapeutic strategies include surgical resection, followed by radiotherapy and chemotherapy. Despite such aggressive multimodal therapy, prognosis remains poor, with a median patient survival of 14 months. A proper understanding of the molecular drivers responsible for GBM progression are therefore necessary to instruct the development of novel targeted agents and enable the design of effective treatment strategies. Activation of the c-Jun N-terminal kinase isoform 2 (JNK2) is reported in primary brain cancers, where it associates with the histologic grade and amplification of the epidermal growth factor receptor (EGFR). In this manuscript, we demonstrate an important role for JNK2 in the tumor promoting an invasive capacity of EGFR variant III, a constitutively active mutant form of the receptor commonly found in GBM. Expression of EGFR variant III induces transactivation of JNK2 in GBM cells, which is required for a tumorigenic phenotype in vivo. Furthermore, JNK2 expression and activity is required to promote increased cellular invasion through stimulation of a hepatocyte growth factor-c-Met signaling circuit, whereby secretion of this extracellular ligand activates the receptor tyrosine kinase in both a cell autonomous and nonautonomous manner. Collectively, these findings demonstrate the cooperative and parallel activation of multiple RTKs in GBM and suggest that the development of selective JNK2 inhibitors could be therapeutically beneficial either as single agents or in combination with inhibitors of EGFR and/or c-Met.

Inhibition of apolipoprotein A-I expression by TNF-alpha in HepG2 cells: requirement for c-jun.

Tumor necrosis factor alpha (TNF α) signals in part through the mitogen activated protein (MAP) kinase c-jun-N-terminal kinase (JNK). Activation of JNK has been shown to promote insulin resistance and dyslipidemia, including reductions in plasma high-density lipoprotein (HDL) and apolipoprotein A-I (apo A-I). To examine how TNF α-mediated JNK activation inhibits hepatic apo A-I production, the effects of c-jun activation on apo A-I gene expression were examined in HepG2 cells. Apo A-I gene expression and promoter activity were measured by Northern and Western blotting and transient transfection. Transient transfection and siRNA were used to specifically over-express or knockout c-jun, c-jun-N-terminal kinase-1 and -2 (JNK1 and JNK2, respectively) and mitogen-activated protein kinase-4 (MKK4). TNF α-treatment of HepG2 cells induced rapid phosphorylation of c-jun on serine 63. In cells treated with phorbol-12-myristate-13-acetate (PMA), apo A-I gene promoter activity was inhibited and apo A-I mRNA content and apo A-I protein secretion decreased. Likewise, over-expression of JNK1 and JNK2 inhibited apo A-I promoter activity. Over-expression of constitutively active MKK4, an upstream protein kinase that directly activates JNK, also inhibited apo A-I promoter activity, while over-expression of a dominant-negative MKK4 de-repressed apo A-I promoter activity in TNF α-treated cells. Inhibition of c-jun synthesis using siRNA but not a control siRNA prevented TNF α-mediated inhibition of apo A-I. These results suggest that the MKK4/JNK/c-jun signaling pathway mediates TNF α-dependent inhibition of apo A-I synthesis.

Roles of phosphorylated JNK in esophageal squamous cell carcinomas of Kazakh ethnic.

The c-Jun NH2 -terminal kinase (JNK) signal pathway has been implicated in the growth, cellular proliferation, and apoptosis in many kinds of carcinomas. However, the role of JNK in the development of esophageal squamous cell carcinomas (ESCCs) is unknown. To investigate the role of JNK in ESCC, in vitro, esophageal cancer cell line Eca109 was pretreated using SP600125, JNK specific inhibitor, then was subjected to MTT assay to examine cellular proliferation, flow cytometric analysis to detect apoptosis and cell cycle, and wound healing assay to evaluate cell migration. Meanwhile, the mRNA and protein expression of JNK in Eca109 cells pretreated with SP600125 were examined by real-time quantitative reverse transcription PCR (qRT-PCR) and Western blotting, respectively. In vivo, 12 paired of fresh ESCC and normal adjacent tissues (NAT) from Kazakh patients were used to validate the expression of JNK by qRT-PCR and Western blotting. Furthermore, to reconfirm the expression trend of activation JNK (p-JNK), enlarged 72 paired of Kazakh's ESCC and NAT were subjected to immunohistochemistry. Our results showed that the suppression of p-JNK could lead to apoptosis and reduce proliferation in Eca109 cells. However, there was an elevated expression of p-JNK protein in NAT compared with ESCC tissues, and there was significant difference between p-JNK expression and pathological differentiation (P < 0.05) in Kazakh populations. Together, all the data we obtained in the present study indicated that the p-JNK MAPK pathway was involved in pathogenesis of Kazakh's ESCC, and played a different roles in carcinogenesis and development of Kazakh's ESCC.

Expression of HSP70 and JNK-related proteins in human liver cancer: Potential effects on clinical outcome.

BACKGROUND: Activation of stress-activated protein kinase/c-Jun N-terminal kinase was inhibited in cells, in which heat shock protein70 was induced to a high level, indicating that heat shock protein70 might be anti-apoptosis protein. AIM: We examined the expression of heat shock protein70 and c-Jun N-terminal kinase signal transduction pathway in human liver carcinoma to explore their relationship and clinical parameters. PATIENTS AND METHODS: The expression of heat shock protein70, c-Jun N-terminal kinase1, c-Jun N-terminal kinase2 and c-Jun were detected immunohistochemically in 62 samples of liver cancer. Western blot was used to confirm immunostaining results. RESULTS: Heat shock protein70 expression showed a positive correlation with the malignant differentiation in liver carcinoma (r=0.449, P<0.0005). The expression of c-Jun N-terminal kinase1, c-Jun N-terminal kinase2, and c-Jun showed a negative correlation with the malignant differentiation in liver carcinoma (r=-0.351, P=0.005; r=-0.303, P=0.017; r=-0.302, P=0.017). Heat shock protein70 expression was correlated with c-Jun N-terminal kinase1 (r=-0.385, P=0.002), c-Jun N-terminal kinase2 (r=-0.309, P=0.015) and c-Jun (r=-0.302, P=0.017). Expression of heat shock protein70, as well as c-Jun N-terminal kinase1, was correlated with recurrence-free survival after the resection. Heat shock protein70 was associated with prognosis (P=0.004). CONCLUSION: Expression of heat shock protein70 and c-Jun N-terminal kinase-related proteins might be an indicator of malignant potential in liver carcinoma. The balance between heat shock protein70 and c-Jun N-terminal kinase-related protein may increase the stability of liver cancer cells in stress. Negative expression of heat shock protein70 might be a protective factor of recurrence of liver carcinoma.

JNK2 and p38 MAPK over-expressions do not represent key events in chronic myeloid leukemia transformation.

Over-expression of two members of MAP kinase family (JNK2 and p38) has been already observed in chronic myeloid leukemia (CML). In the present study, significance of this deregulation was investigated. Impacts of JNK2/p38 suppression on gene expression profile of CML cell lines (K562/KU-812) were studied using an experimental approach that combines siRNA-mediated specific inhibition of the genes and array-based expression analyses. After JNK2 depletion, 27 out of 588 tested genes showed significant expression changes, with 13 down-regulated genes and 14 up-regulated genes. Among others, expression of MSH2 and MSH6, mdm2, and caspase-2 was reduced and, on the other hand, MKK1 and MKK6, RFC2, cytokeratins K18 and K19, BAD, and DR5 expression was up-regulated. In the case of p38 silencing, 20 genes were considered as significantly deregulated (7 genes reduced, 13 over-expressed). These genes included caspase-10, SOD1, and Notch4 (down-regulation) and caspase-2 and caspase-3, CDC2, CDK4, and c-kit (up-regulation). In conclusion, comparison of expression profiles after JNK2 or p38 gene silencing revealed distinct sets of affected genes. The results implied an unequal impact of the MAPK deregulation on the CML cells. Further, we demonstrated that neither JNK2 nor p38 siRNAmediated inhibition led to significant change of CML cell proliferation. It suggests that there are other important, likely upstream regulators essential for CML malignant cell growth/transformation; therefore, separate inhibition of JNK2 or p38 MAPK gene is not sufficient for a proliferation arrest.

Phloretin induces apoptosis of non-small cell lung carcinoma A549 cells via JNK1/2 and p38 MAPK pathways.

Phloretin (Ph) existing in apples, pears and various vegetables is known to have antitumor activities in several cancer cell lines. However, little is known about its effect on human lung cancer cells. The aim of the present study was to see whether Ph could induce apoptosis of non-small cell lung cancer (NSCLC) cells, and explore the possible underlying mechanism of action. We found that Ph markedly induced cell apoptosis of NSCLC cell line A549, and inhibited the migration of A549 cells in a dose-dependent manner. The expression level of BAX, cleaved caspase-3 and -9, and degraded form of PARP was increased and Bcl-2 was decreased after Ph treatment. In addition, the phosphorylation of P38 MAPK, ERK1/2 and JNK1/2 was increased in a dose­dependent manner in parallel with Ph treatment. Inhibition of P38 MAPK and JNK1/2 by specific inhibitors significantly abolished the Ph-induced activation of the caspase-3 and -9. In vivo tumor-suppression assay further indicated that Ph (20 mg/kg) displayed a more significant inhibitory effect on A549 xenografts in tumor growth. All these findings indicate that Ph is able to inhibit NSCLC A549 cell growth by inducing apoptosis through P38 MAPK and JNK1/2 pathways, and therefore may prove to be an adjuvant to the treatment of NSCLC.

A critical role for MAP kinases in the control of Ah receptor complex activity.

The heterodimeric complex of aromatic hydrocarbon receptor (AHR) and Ah receptor nuclear translocator (ARNT) plays a pivotal role in controlling the expression of drug metabolism genes, such as the cytochromes p450 (Cyp) 1a1 and 1b1, believed to be responsible for most toxic effects of the environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). In this study, we show that activation of Jun N-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK) modulates ARNT transcription activity and potentiates the transcriptional activity of AHR/ARNT complexes. Inhibition of ERK by chemical compounds and ablation of JNK caused significant decreases in CYP1A1 induction by TCDD. Compared to wild type, JNK2 ablation significantly reduced TCDD-stimulated CYP1A1 expression in mouse thymus and testis, but not in liver. In contrast, CYP1B1 expression was unaffected in all three tissues of the knockout mice. These data suggest that JNK and ERK modulate ARNT activity and AHR/ARNT-dependent gene expression, contributing to the gene-specific and tissue-specific toxicity of environmental contaminants.

CIP2A signature reveals the MYC dependency of CIP2A-regulated phenotypes and its clinical association with breast cancer subtypes.

Protein phosphatase 2A (PP2A) is a critical human tumor-suppressor complex. A recently characterized PP2A inhibitor protein, namely cancerous inhibitor of PP2A (CIP2A), has been found to be overexpressed at a high frequency in most of the human cancer types. However, our understanding of gene expression programs regulated by CIP2A is almost absent. Moreover, clinical relevance of the CIP2A-regulated transcriptome has not been addressed thus far. Here, we report a high-confidence transcriptional signature regulated by CIP2A. Bioinformatic pathway analysis of the CIP2A signature revealed that CIP2A regulates several MYC-dependent and MYC-independent gene programs. With regard to MYC-independent signaling, JNK2 expression and transwell migration were inhibited by CIP2A depletion, whereas MYC depletion did not affect either of these phenotypes. Instead, depletion of either CIP2A or MYC inhibited cancer cell colony growth with statistically indistinguishable efficiency. Moreover, CIP2A depletion was shown to regulate the expression of several established MYC target genes, out of which most were MYC-repressed genes. CIP2A small-interfering RNA-elicited inhibition of colony growth or activation of MYC-repressed genes was reversed at large by concomitant PP2A inhibition. Finally, the CIP2A signature was shown to cluster with basal-type and human epidermal growth factor receptor (HER)2-positive (HER2+) breast cancer signatures. Accordingly, CIP2A protein expression was significantly associated with basal-like (P=0.0014) and HER2+ (P<0.0001) breast cancers. CIP2A expression also associated with MYC gene amplification (P<0.001). Taken together, identification of CIP2A-driven transcriptional signature, and especially novel MYC-independent signaling programs regulated by CIP2A, provides important resource for understanding CIP2A's role as a clinically relevant human oncoprotein. With regard to MYC, these results both validate CIP2A's role in regulating MYC-mediated gene expression and provide a plausible novel explanation for the high MYC activity in basal-like and HER2+ breast cancers.

Knockdown of the c-Jun-N-terminal kinase expression by siRNA inhibits MCF-7 breast carcinoma cell line growth.

We have examined the effect of two small interference RNA against Jnk-1 and Jnk-2 in the breast cancer cell line MCF-7. The expression of the JNK-1 and JNK-2 is frequently elevated in breast cancer and is a frequent genetic abnormality in this malignancy. For a better understanding of its role in maintaining the malignant phenotype, we used small RNA interference (siRNA) directed against Jnk-1 or Jnk-2. We made control and Jnk-1 and Jnk-2 siRNA using vector plasmid, which was then transfected to reduce its expression in MCF-7 cells. We assessed the effects of JNK-1 or JNK-2 silencing on cell growth by western blot analysis, soft agar assay, cell proliferation assay, cell viability by MTT assay and caspase assay in vitro. Our data showed that siRNA against Jnk-1 or Jnk-2 markedly and durably reduced its expression in MCF-7 cells by up to 70%, decreased the growth rate of MCF-7 cells, inhibited colony formation in soft agar and significantly reduced cell growth in MCF-7 carcinoma culture cell line. We also found that depletion of JNK-1/2 in this manner promoted apoptosis of MCF-7 cells upon serum withdrawal. In addition, we found that MCF-7 cells did not exhibit any caspase-3 activity. In conclusion, we observed that JNK-1 and JNK-2 have a pivotal function in the development of breast cancer. Our data show that decreasing the JNK-1 or JNK-2 protein level in MCF-7 cells by siRNA could significantly inhibit MCF-7 cell growth in in vitro assay, and imply the therapeutic potential of siRNA on the treatment of breast cancer by targeting overexpression kinases such as JNK-1/2 and might be a potential therapeutic target for human breast cancer.

Anti-DR5 mAb ameliorate adjuvant arthritis rats through inducing synovial cells apoptosis.

OBJECTIVE: Study the therapeutic effects and immunoregulatory mechanisms of anti-DR5 mAb on adjuvant arthritis (AA) rats. METHODS: AA rats induced by CFA, were treated with anti-DR5 mAb through mainline administration. Effect on the synovial membranes of the tissues was detected by H&E staining. Flow cytometry and MTT assay were used for detecting the induced apoptosis in an in vitro system and TUNEL assay was used for analysis in an in vivo system. The involvement of the apoptotic pathway was further proved by a caspase inhibition assay. RESULTS: Anti-DR5 mAb could induce synovial cell apoptosis in an in vitro system, which was related with the mRNA expression of DR5 on the cell surface. The mRNA expressions of c-myc and bcl-2 were decreased in synovial cells and those of p21, p53, and bax were increased. The protein expressions of caspase-8/3/9, RANKL, JNK2, and c-Jun were raised and that of bcl-2 was decreased. When the caspase inhibitor was added to the synovial cells treated with anti-DR5 mAb, it showed a dose-dependence inhibition effect, indicating that anti-DR5 mAb inducing apoptosis might be through the caspase pathway. CONCLUSION: This study shows that anti-DR5 mAb can ameliorate arthritic symptoms. The mechanisms of the treatment are related to the increase in synovial cell apoptosis by regulating the mRNA expression of DR5 and apoptosis-related genes, prolonging the duration of the cell cycle by modulation of the mRNA expression of cell cycle-related genes, and the protein expression of the molecules in the caspase pathway and RANKL, JNK2, and c-Jun.

Decreased expression of phosphorylated JNK in breast infiltrating ductal carcinoma is associated with a better overall survival.

Phosphorylation/activation of c-jun NH2-terminal kinase (JNK) has an ambivalent role, pro-proliferation or antiproliferation, in human cancers, which is determined by different cell types and by its crosstalk with other kinases. So far, the role of phosphorylated JNK (p-JNK) in breast cancer is mostly undefined. In this study, we analyzed the expression of p-JNK, as well as p-ERK1/2 and p-38, in the pair of cancer and noncancer breast tissues, by using immunoblotting techniques. These results were further correlated with the clinicopathological characteristics and overall survival. Decreased p-JNK1/2 expression in cancer tissues was observed in 48.5% of breast infiltrating ductal carcinoma (IDC) cases and was correlated significantly with the increased tumor grade and the decreased age at diagnosis (p = 0.030 and 0.029). Interestingly, the Kaplan-Meier survival curve showed that the decreased p-JNK1/2 expression was associated with a better overall survival of IDC (p = 0.004). The expression of p-JNK1/2 was positively correlated with p-p38 (p = 0.002), but not p-ERK1/2. Furthermore, co-expressed p-JNK1/2 and p-p38 was associated with a poor overall survival of IDC (p = 0.007). In conclusion, our results indicate that the aberrant p-JNK1/2 expression and the co-expressed p-JNK1/2 and p-p38 in breast tissues may play a role in the carcinogenesis of breast IDC.

Impaired long-term potentiation in c-Jun N-terminal kinase 2-deficient mice.

c-Jun N-terminal kinases (JNKs) are thought to be involved in regulating synaptic plasticity. We therefore investigated the specific role of JNK2 in modulating long-term potentiation (LTP) in hippocampus during development, using JNK2-deficient mice. The morphological structure and the numbers of both NeuN, a specific neuronal marker, and GABA-positive neurons in the hippocampal areas were similar in wild-type and Jnk2(-/-) mice. Western blot analysis revealed that JNK2 expression was higher and stable at 1 and 3 months of age, but JNK1 levels were lower at 1 month of age and almost undetectable in 3-month-old wild-type mice. In contrast to wild-type mice, there was a significant increase in JNK1 expression in JNK2 mutant mice, especially at 1 month of age. Electrophysiological studies demonstrated that LTP was impaired in both the CA1 and CA3 regions in 1-month-old, but not in adult, Jnk2(-/-) mice, probably owing to decreased presynaptic neurotransmitter release. Moreover, late-phase LTP, but not early-phase LTP, was impaired in the Jnk2(-/-) adult mice, suggesting that JNK2 plays a role in transforming early LTP to late LTP. Together, the data highlight the specific role of JNK2 in hippocampal synaptic plasticity during development.

JNK/ERK-AP-1/Runx2 induction "paves the way" to cartilage load-ignited chondroblastic differentiation.

Chondro-osteogenesis and subsequently skeletal morphology are greatly influenced by mechanical loads. The exact mechanism(s) by which mechanical stimuli are transduced in chondrocytes remains obscure and appears to be equally complex with similar signal transducing systems. Here we investigated whether and to what extent the MAPK (JNK/ERK)-AP-1/Runx2 signaling pathways are engaged in this phenomenon, and assessed their involvement in the functional biology of articular cartilage. For this purpose, 14-day-old female Wistar rats were divided into 2 groups: the first group was fed hard diet (simulating physiologic temporomandibular joint (TMJ) loading), while the second group was fed soft diet (reduced TMJ loading). On day 21 (experiment initiation day - weaning day), biopsies from condyles of both groups were obtained after 6, 12 and 48 h of functional TMJ loading. Immunohistochemical methodology was employed to evaluate the expression levels of pc-Jun, c-Fos, JNK2, p-JNK, p-ERK and Runx2 due to alteration in functional load. Our data demsonstrate that the protein levels of all the aforementioned molecules were markedly increased in animals fed with the hard diet, throughout the experimental procedure. These results indicate that functional cartilage loading induces the AP-1 and Runx2 transcription factors through the JNK and ERK MAPK cascades. In as much as the above signaling mediators/effectors are considered to be crucial in the differentiation/maturation process of cartilage tissue, we pose that functional mechanical loading of condylar cartilage serves to "fine tune" chondroblastic differentiation/maturation.

Neuroprotective effects of preconditioning ischemia on ischemic brain injury through down-regulating activation of JNK1/2 via N-methyl-D-aspartate receptor-mediated Akt1 activation.

Our previous studies have demonstrated that the JNK signaling pathway plays an important role in ischemic brain injury and is mediated via glutamate receptor 6. Others studies have shown that N-methyl-d-aspartate (NMDA) receptor is involved in the neuroprotection of ischemic preconditioning. Here we examined whether ischemic preconditioning down-regulates activation of the mixed lineage kinase-JNK signaling pathway via NMDA receptor-mediated Akt1 activation. In our present results, ischemic preconditioning could not only inhibit activations of mixed lineage kinase 3, JNK1/2, and c-Jun but also enhanced activation of Akt1. In addition, both NMDA (an agonist of NMDA receptor) and preconditioning showed neuroprotective effects. In contrast, ketamine, an antagonist of NMDA receptor, prevented the above effects of preconditioning. Further studies indicated that LY294002, an inhibitor of phosphoinositide 3-kinase that is an upstream signaling protein of Akt1, could block neuroprotection of preconditioning, and KN62, an inhibitor of calmodulin-dependent protein kinase, also achieved the same effects as LY294002. Therefore, both phosphoinositide 3-kinase and calmodulin-dependent protein kinase are involved in the activation of Akt1 in ischemic tolerance. Taken together, our results indicate that preconditioning can inhibit activation of JNK signaling pathway via NMDA receptor-mediated Akt1 activation and induce neuroprotection in hippocampal CA1 region.

Expression of stress-activated kinases c-Jun N-terminal kinase (SAPK/JNK-P) and p38 kinase (p38-P), and tau hyperphosphorylation in neurites surrounding betaA plaques in APP Tg2576 mice.

Hyperphosphorylated tau in neurites surrounding beta-amyloid (betaA) deposits, as revealed with phospho-specific anti-tau antibodies, are found in amyloid precursor protein (APP) Tg2576 mice. Because betaA is a source of oxidative stress and may be toxic for cultured cells, the present study examines the expression of phosphorylated (active) stress-activated kinase c-Jun N-terminal kinase (SAPK/JNK-P) and p38 kinase (p38-P), which have the capacity to phosphorylate tau at specific sites, and their specific substrates c-Jun and ATF-2, which are involved in cell death and survival in several paradigms, in Tg2576 mice. The study was planned to shed light about the involvement of these kinases in tau phosphorylation in cell processes surrounding amyloid plaques, as well as in the possible phosphorylation (activation) of c-Jun and activating transcription factor-2 (ATF-2) in relation to betaA deposition. Moderate increase in the expression of phosphorylated mitogen-activated protein kinase and extracelullar signal-regulated kinase (MAPK/ERK-P) occurs in a few amyloid plaques. However, strong expression of SAPK/JNK-P and p38-P is found in the majority of, if not all, amyloid plaques, as seen in serial consecutive sections stained for betaA and stress kinases. Moreover, confocal microscopy reveals colocalization of phospho-tau and SAPK/JNK-P, and phospho-tau and p38-P in many dystrophic neurites surrounding amyloid plaques. Increased expression levels of nonbound tau, SAPK/JNK-P and p38-P are corroborated by Western blots of total cortical homogenate supernatants in Tg2576 mice when compared with age-matched controls. No increase in phosphorylated c-JunSer63 (c-Jun-P) and ATF-2Thr71 (ATF-2-P) is found in association with betaA deposits. In addition, no expression of active (cleaved) caspase-3 (17 kDa) has been found in transgenic mice. Taken together, these observations provide a link between betaA-induced oxidative stress, activation of stress kinases SAPK/JNK and p38, and tau hyperphosphorylation in neurites surrounding amyloid plaques, but activation of these kinases is not associated with accumulation of c-Jun-P and ATF-2-P, nor with activation of active caspase-3 in the vicinity of betaA deposits.