Lack of p38 MAP kinase activation in TRAIL-resistant cells is not related to the resistance to TRAIL-mediated cell death

Activation of MAP kinases is involved in various cellular processes, including immunoregulation, inflammation, cell growth, cell differentiation, and cell death. To investigate the role of p38 MAP kinase activation in the signaling pathway of TRAIL-mediated apoptosis, we compared TRAIL-mediated MAP kinase activation in TRAIL-susceptible human colon cancer cell line DLD1 and TRAIL-resistant DLD1/TRAIL-R cells. TRAIL-mediated activation of ERK occurred in both cell lines. In contrast, both DLD1 and DLD1/TRAIL-R cells showed no obvious JNK activation after treatment with TRAIL. Interestingly, TRAIL-mediated activation of p38 MAP kinases was observed in DLD1 cells but not in DLD1/TRAIL-R cells. However, activation of p38 MAP kinases was observed in both DLD1 and DLD1/TRAIL-R cells after treatment with anisomycin. Furthermore, inhibiting activated p38 MAP kinases with known inhibitors or with an adenovector expressing dominant negative p38alpha did not block TRAIL-mediated cell death in DLD1 cells. Moreover, activation of p38 MAP kinases by adenovectors expressing constitutive MKK3 or MKK6 (Ad/MKK3bE or Ad/MKK6bE) did not induce cell death in either DLD1 or DLD1/TRAIL-R cell lines. Our results suggest that activation of p38 MAP kinases does not play a major role in TRAIL-mediated apoptosis in DLD1 cells and that lack of TRAIL-mediated p38 MAP kinase activation may not be the mechanism of TRAIL-resistance in DLD1/TRAIL-R cells.     

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.     

c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK) is required for mitoxantrone- and anisomycin-induced apoptosis in HL-60 cells.

Programmed cell death, or apoptosis, has emerged as a common mechanism by which cells respond to chemotherapeutic drugs. However, the signaling mechanisms that mediate drug-induced apoptosis are still widely unknown. Mitogen-activated protein kinase (MAPK) signaling cascades trigger stimulus-specific responses in cells with ERK being associated with proliferation and differentiation, and JNK/SAPK and p38 mediating stress and apoptotic responses. Here, we found that mitoxantrone and anisomycin stimulated a dose- and time-dependent induction of JNK/SAPK activity, and to a lesser extent p38 activity, that preceded the appearance of apoptosis as measured by internucleosomal DNA fragmentation. These compounds did not induce ERK activity. We further demonstrated that p38 activity was not involved in the induction of apoptosis since the use of the p38 inhibitor, SB203580, did not prevent drug-induced apoptotic DNA fragmentation. Additionally, direct inhibition of JNK/SAPK signaling through the use of dominant-negative MKK4/SEK1 (SEK-AL) inhibited mitoxantrone- and anisomycin-induced apoptosis. These results suggest that mitoxantrone- and anisomycin-induced apoptosis is dependent on JNK/SAPK, but not p38, activity.     

Defining MAP3 kinases required for MDA-MB-231 cell tumor growth and metastasis

Analysis of patient tumors suggests that multiple MAP3 kinases (MAP3Ks) are critical for growth and metastasis of cancer cells. MAP3Ks selectively control the activation of extracellular signal-regulated kinase 1/2 (ERK1/2), Jun N-terminal kinase (JNK), p38 and ERK5 in response to receptor tyrosine kinases and GTPases. We used MDA-MB-231 cells because of their ability to metastasize from the breast fat pad to distant lymph nodes for an orthotopic xenograft model to screen the function of seven MAP3Ks in controlling tumor growth and metastasis. Stable short hairpin RNA (shRNA) knockdown was used to inhibit the expression of each of the seven MAP3Ks, which were selected for their differential regulation of the MAPK network. The screen identified two MAP3Ks, MEKK2 and MLK3, whose shRNA knockdown caused significant inhibition of both tumor growth and metastasis. Neither MEKK2 nor MLK3 have been previously shown to regulate tumor growth and metastasis in vivo. These results demonstrated that MAP3Ks, which differentially activate JNK, p38 and ERK5, are necessary for xenograft tumor growth and metastasis of MDA-MB-231 tumors. The requirement for MAP3Ks signaling through multiple MAPK pathways explains why several members of the MAPK network are activated in cancer. MEKK2 was required for epidermal growth factor receptor and Her2/Neu activation of ERK5, with ERK5 being required for metastasis. Loss of MLK3 expression increased mitotic infidelity and apoptosis in vitro. Knockdown of MEKK2 and MLK3 resulted in increased apoptosis in orthotopic xenografts relative to control tumors in mice, inhibiting both tumor growth and metastasis; MEKK2 and MLK3 represent untargeted kinases in tumor biology for potential therapeutic development.     

Role of mitogen-activated protein kinase kinase 4 in cancer

Mitogen-activated protein (MAP) kinase kinase 4 (MKK4) is a component of stress activated MAP kinase signaling modules. It directly phosphorylates and activates the c-Jun N-terminal kinase (JNK) and p38 families of MAP kinases in response to environmental stress, pro-inflammatory cytokines and developmental cues. MKK4 is ubiquitously expressed and the targeted deletion of the Mkk4 gene in mice results in early embryonic lethality. Further studies in mice have indicated a role for MKK4 in liver formation, the immune system and cardiac hypertrophy. In humans, it is reported that loss of function mutations in the MKK4 gene are found in approximately 5% of tumors from a variety of tissues, suggesting it may have a tumor suppression function. Furthermore, MKK4 has been identified as a suppressor of metastasis of prostate and ovarian cancers. However, the role of MKK4 in cancer development appears complex as other studies support a pro-oncogenic role for MKK4 and JNK. Here we review the biochemical and functional properties of MKK4 and discuss the likely mechanisms by which it may regulate the steps leading to the formation of cancers.     

Differential regulation of Jun N-terminal kinase and p38MAP kinase by Galpha12

Based on the findings that the overexpression of the wild-type Galpha(12) (Galpha(12)WT) result in the oncogenic transformation of NIH3T3 cells in a serum-dependent manner, a model system has been established in which the mitogenic and subsequent cell transformation pathways activated by Galpha(12) can be turned on or off by the addition or removal of serum. Using this model system, our previous studies have shown that the stimulation of Galpha(12)WT or the expression of an activated mutant of Galpha(12) (Galpha(12)QL) leads to increased cell proliferation and subsequent oncogenic transformation of NIH3T3 cells, as well as persistent activation of Jun N-terminal kinases (JNKs). In the present studies, we show that the stimulation of Galpha(12)WT or the expression of Galpha(12)QL results in a potent inhibition of p38MAPK, and that the mechanism by which Galpha(12) inhibits p38MAPK activity involves the dual specificity kinases upstream of p38MAPK. The results indicate that Galpha(12) attenuates the activation of MKK3 and MKK4, which are known to stimulate only p38MAPK or p38MAPK and JNK, respectively. The results also suggest that Galpha(12) activates JNKs specifically through the stimulation of the JNK-specific upstream kinase MKK7. These findings demonstrate for the first time that Galpha(12) differentially regulates JNK and p38MAPK by specifically activating MKK7, while inhibiting MKK3 and MKK4 in NIH3T3 cells. Since the stimulation of p38MAPK is often associated with apoptotic responses, our findings suggest that Galpha(12) stimulates cell proliferation and neoplastic transformation of NIH3T3 cells by attenuating p38MAPK-associated apoptotic responses, while activating the mitogenic responses through the stimulation of ERK- and JNK-mediated signaling pathways.     

New paradigms for the function of JNKK1/MKK4 in controlling growth of disseminated cancer cells

Much work has been done in the 20 years since the discovery of the first metastasis suppressor gene to investigate the diverse biochemical functions of the proteins these genes encode. The function of metastasis suppressors cannot be solely predicted from correlative clinical data or in vitro studies. Instead, careful design of in vivo experiments to test broader hypotheses is necessary to pinpoint the mechanism of action of these novel proteins. Our laboratory identified c-Jun NH2-terminal kinase activating kinase 1 (JNKK1)/Mitogen-activated protein kinase (MAPK) kinase 4 (JNKK1/MKK4) as a metastasis suppressor in prostate and ovarian cancer. JNKK1/MKK4 is a stress activated protein kinase (SAPK) involved in a variety of signaling events, ranging from the regulation of hepatoblast survival during mammalian development to metastasis suppression in adult ovarian and prostate cancers. JNKK1/MKK4 function has typically been associated with the c-Jun NH2-terminal kinase (JNK) signaling pathway, particularly in the immune system where JNK plays a role in inflammatory signaling and apoptosis. However, evidence continues to accumulate that JNKK1/MKK4 is also a physiologic activator of p38 under certain conditions, and that activation of p38 arrests cell cycle progression. This review will provide a historical perspective on the role of JNKK1/MKK4 in SAPK signaling, including some recent findings from our own laboratory that shed light on the complicated role for JNKK1/MKK4 in metastatic colonization.     

N-(4-hydroxyphenyl)retinamide-induced apoptosis triggered by reactive oxygen species is mediated by activation of MAPKs in head and neck squamous carcinoma cells

N-(4-hydroxyphenyl)retinamide (4HPR), a synthetic retinoid effective in cancer chemoprevention and therapy, is thought to act via apoptosis induction resulting from increased reactive oxygen species (ROS) generation. As ROS can activate MAP kinases and protein kinase C (PKC), we examined the role of such enzymes in 4HPR-induced apoptosis in HNSCC UMSCC22B cells. 4HPR increased ROS level within 1 h and induced activation of caspase 3 and PARP cleavage within 24 h. Activation of MKK3/6 and MKK4, JNK, p38 and ERK was detected between 6 and 12 h, increased up to 24 h and preceded apoptosis. 4HPR-induced activation of these kinases was abrogated by the antioxidants BHA and vitamin C. SP600125, a JNK inhibitor, suppressed 4HPR-induced c-Jun phosphorylation, cytochrome c release from mitochondria and apoptosis. Suppression of JNK1 and JNK2 using siRNA decreased, whereas overexpression of wild type-JNK1 enhanced 4HPR-induced apoptosis. PD169316, a p38, inhibitor suppressed phosphorylation of Hsp27 and apoptosis. PD98059, an MEK1/2 inhibitor, also suppressed ERK1/2 activation and apoptosis induced by 4HPR. Likewise, PKC inhibitor GF109203X suppressed ERK and p38 phosphorylation and PARP cleavage. These data indicate that 4HPR-induced apoptosis is triggered by ROS increase, leading to the activation of the mitogen-activated protein serine/threonine kinases JNK, p38, PKC and ERK, and subsequent apoptosis.     

The PI 3-kinase-Rac-p38 MAP kinase pathway is involved in the formation of signet-ring cell carcinoma

Signet-ring cell carcinoma is classified in poorly differentiated adenocarcinoma with an aggressive nature and a poor prognosis. We have shown that the activation of PI 3-kinase in highly differentiated adenocarcinomas induces loss of cell-cell contact and formation of vacuoles, giving phenotypes similar to those of signet-ring cell lines. SB203580, a potent p38 MAP kinase inhibitor, blocked this transition, and expression of an active form of MKK6 (MKK6DA), an activator of p38 MAP kinase, gave effects similar to those induced by expression of the active form of PI 3-kinase (BD110), although formation of large vacuoles was not induced. Activation of MKK3, another activator of p38 MAP kinase, was activated in native signet-ring carcinoma cell lines. Anchorage-independent growth of signet-ring cell lines was inhibited by LY294002 or SB203580. These results suggest that p38 MAP kinase is functioning downstream of PI 3-kinase in signaling of the malignant phenotype. Secretion of mucins was enhanced in BD110-expressing cells, but not in MKK6DA-expressing cells, suggesting that secretion of mucins is independent of the MKK6-p38 MAP kinase cascade. Thus, there may be at least two pathways, p38 MAP kinase-dependent and -independent, which are involved in regulation of cell-cell contact and the protein secretion system, respectively.     

The oncogenic TEL/PDGFR beta fusion protein induces cell death through JNK/SAPK pathway.

The TEL/PDGFR beta (T/P) fusion protein isolated from patients bearing a t(5;12) translocation is transforming when expressed in haematopoietic cells. To examine the signal transduction events activated by this protein, we measured the effect of T/P on activation of the c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK) in mouse bone marrow-derived Ba/F3 cells. Significant increase in the activity of JNK/SAPK1 was observed in transient transfection as well as in Ba/F3 cells stably expressing T/P. This activation was abrogated when the T/P-expressing cells were treated with a specific inhibitor of the PDGFR beta tyrosine kinase, indicating that the activity of the PDGFR beta part of the fusion protein was involved in JNK/SAPK activation. Expression of a dominant negative mutant of mitogen-activated protein kinase kinase 4 (MKK4), a direct activator of JNK/SAPK, prevented T/P-induced JNK/SAPK activation. In addition, inhibition of phosphoinositide-3 OH kinase (PI-3 kinase), a promoting survival factor, potentiated the effect of T/P on JNK/SAPK activation. Interestingly, expression of T/P was shown to initiate an apoptotic response that was enhanced by treatment of cells with the PI-3 kinase inhibitor LY294002, suggesting that T/P mediated cell death through activation of JNK/SAPK signalling pathway. Consistent with this hypothesis, expression of the dominant negative mutant of MKK4 decreased T/P-mediated apoptosis, while a dominant-negative mutant of PI-3 kinase enhances cell death. These findings indicate that activation of JNK/SAPK by T/P is related to apoptosis rather than cell proliferation and transformation.     

Critical role of the c-JunNH2-terminal kinase and p38 mitogen-activated protein kinase pathways on sodium butyrate-induced apoptosis in DU145 human prostate cancer cells.

Sodium butyrate (NaBu) is known to exhibit anti-cancer effects via the differentiation and apoptosis of various carcinoma cells. However, the mechanism by which NaBu induces apoptosis and the involvement of protein kinases during apoptosis is not completely understood. To investigate the underlying pathways, we performed cell culture experiments in androgen-independent human prostate cancer (DU145 cells) focusing on various protein kinases. NaBu causes concentration-dependent cell detachment and growth inhibition. Exposure of DU145 cells to NaBu for 24 h caused a strong apoptotic effect with 26% nuclear fragmentation and condensation. In addition, NaBu induced caspase-3 and poly-ADP ribose polymerase cleavage and up-regulation of bax, suggesting that mitochondrial damage is involved in NaBu-induced caspase-dependent apoptosis. Interestingly, NaBu stimulated p38 mitogen-activated protein kinase (MAPK) and c-Jun NH2-terminal kinase (JNK) activation, but not extracellular signal-regulated kinase 1/2 activation during apoptosis. Furthermore, NaBu up-regulated total protein levels and phospho forms of MAPK kinase 3 (MKK3) and MAPK kinase 4 (MKK4) as the upstream kinases of p38 MAPK and JNK independently of oxidative stress. Taken together, it is suggested that NaBu can be a promising chemopreventive agent for prostate cancer and the p38 MAPK and JNK pathways have critical roles in NaBu-induced apoptosis in DU145 cells.     

From receptors to stress-activated MAP kinases

The cell signaling pathways that culminate in activation of a family of stress-activated MAP kinases are beginning to be defined. Determination of cell life and cell death is known to largely depend on the balance of intrinsic life and death signals within cells. Recently, two representative mammalian stress-activated kinases, the JNK and p38 MAP kinases, have been implicated in determination of cell fate by modifying the life, death and differentiation signals. However, the molecular mechanisms by which extracellular signals are transmitted from membrane receptors to the most upstream kinases in the JNK and p38 signaling modules are not fully understood. This review will provide an overview of current knowledge of molecular links between inflammatory cytokine receptors and stress-activated MAP kinase cascades.     

Role of MAP kinase signalling pathways in the mode of action of peroxisome proliferators

Peroxisome proliferators (PPs) are a class of non-genotoxic chemicals that cause rodent liver enlargement and hepatocarcinogenesis. In primary rat hepatocytes, PPs cause cell proliferation, suppression of apoptosis and peroxisome proliferation. We have investigated the role of different families of mitogen-activated protein (MAP) kinases in the mode of action of PPs. Addition of 50 microM nafenopin to primary rat hepatocyte cultures caused weak activation of extracellular signal regulated kinases and p38 MAP kinase. However, incubation of primary hepatocytes with the p38 MAP kinase inhibitor SB203580 or the MAP kinase kinase (MEK) inhibitor PD098059 prevented the induction of DNA synthesis and the suppression of transforming growth factor beta(1)-induced apoptosis by the PP nafenopin. In contrast, in the presence of these MAP kinase inhibitors, nafenopin still induced palmitoyl CoA oxidation, a measure of peroxisome proliferation. We have shown previously that PPs such as nafenopin require tumour necrosis factor alpha (TNF-alpha) to exert their effects on cellular proliferation and apoptosis. Here we show that treatment of primary rat hepatocyte cultures with nafenopin causes an increase in bioactive TNF-alpha and that this process requires p38 MAP kinase activity.     

The p38 kinases MKK4 and MKK6 suppress metastatic colonization in human ovarian carcinoma

Despite considerable efforts to improve early detection of ovarian cancer, the majority of women at time of diagnosis will have metastatic disease. Understanding and targeting the molecular underpinnings of metastasis continues to be the principal challenge in the clinical management of ovarian cancer. Whereas the multistep process of metastasis development has been well established in both clinical and experimental models, the molecular factors and signaling pathways involved in successful colonization of a secondary site by disseminated cancer cells are not well defined. We have previously identified mitogen-activated protein kinase (MAPK) kinase 4/c-Jun NH2-terminal kinase (JNK)-activating kinase (MKK4/JNKK1/SEK1, hereafter referred to as MKK4) as a metastasis suppressor protein in ovarian carcinoma. In this study, we elucidate key mechanisms of MKK4-mediated metastasis suppression. Through the use of a kinase-inactive mutant, we show that MKK4 kinase activity is essential for metastasis suppression and prolongation of animal survival. Because MKK4 can activate either of two MAPKs, p38 or JNK, we expressed MKK6 or MKK7, specific activators of these MAPKs, respectively, to delineate which MAPK signaling module was involved in MKK4-mediated metastasis suppression. We observed that MKK6 expression suppressed metastatic colonization whereas MKK7 had no effect. Our finding that MKK4 and MKK6 both suppress metastasis points to the p38 pathway as an important regulatory pathway for metastatic colonization in ovarian cancer.     

MAP kinase pathways involved in glioblastoma response to erucylphosphocholine

Erucylphosphocholine (ErPC) is a promising antineoplastic drug for the treatment of malignant brain tumors. It exerts strong anticancer activity and induces apoptosis even in chemoresistant glioma cells. In the present study, A172 and U373MG glioma cells were treated with ErPC to explore the contribution of MAP kinase family members ERK, JNK and p38 kinase to ErPC-induced cell death. The exposure to ErPC led to activation of JNK and concurrent inhibition of ERK in both cell lines. Using specific MAP kinase inhibitors we confirmed that in U373MG cells ERK was blocked and JNK was activated upon ErPC treatment. Both effects were dependent on caspase activation. In A172 cells, ErPC treatment resulted in an activation of the JNK pathway, whereas the situation with respect to ERK signalling was more complex. We conclude that inhibition of the ERK pathway by ErPC may be related to antiproliferative effects, while activation of the JNK pathway may be responsible for its pro-apoptotic action.