12-lipoxygenase metabolite 12(S)-HETE stimulates human pancreatic cancer cell proliferation via protein tyrosine phosphorylation and ERK activation.

We previously reported that inhibition of the 12-lipoxygenase pathway abolished proliferation and induced apoptosis in several pancreatic cancer cell lines. Furthermore, the 12-lipoxygenase product 12(S)-HETE stimulated pancreatic cancer cell proliferation and reversed 12-lipoxygenase inhibitor-induced growth inhibition. We investigated the underlying mechanism for 12(S)-HETE-induced pancreatic cancer cell proliferation, using 2 human pancreatic cancer cell lines, PANC-1 and HPAF. Cell proliferation was monitored by both thymidine incorporation and cell number. Western blotting was used to investigate the effect of 12(S)-HETE on cellular protein tyrosine phosphorylation as well as ERK, P38 MAPK and JNK/SAPK phosphorylation. 12(S)-HETE markedly stimulated proliferation of pancreatic cancer cells in a time- and concentration-dependent manner. In parallel, 12(S)-HETE induced tyrosine phosphorylation of multiple cellular proteins, while inhibition of tyrosine kinase by genestein abolished 12(S)-HETE-induced proliferation, indicating that intracellular protein tyrosine kinase activation is involved in the mitogenic effects of 12(S)-HETE. Following treatment with 12(S)-HETE, both ERK and P38 MAPK, but not JNK/SAPK, were phosphorylated. The specific MEK inhibitors PD098059 and U0126, which in turn suppress ERK, abolished 12(S)-HETE-stimulated proliferation. In contrast, inhibition of P38 MAPK with SB203580 did not affect 12(S)-HETE-stimulated pancreatic cancer cell proliferation. Furthermore, 12(S)-HETE-stimulated ERK phosphorylation was inhibited by genestein, indicating that tyrosine phosphorylation is essential for ERK activation. These findings suggest that both ERK and cellular protein tyrosine kinase activation are involved in 12(S)-HETE-induced pancreatic cancer cell proliferation but P38 and JNK/SAPK are not involved in this mitogenic effect.     

p38 MAP kinase controls EGF receptor downregulation via phosphorylation at Ser1046/1047

The desensitization mechanism of the EGF receptor (EGFR) is important for the regulation of cancer cells. Although the phosphorylation of EGFR at Tyr1045 and Ser1046/1047 (Ser1046/7) reportedly accounts for such desensitization, the precise mechanism still remains unknown. Therefore, the present study investigated the upstream signals of these phosphorylations in SW480 colon cancer cells. Anisomycin, a potent kinase activator, induced the activation of both p38 mitogen-activated protein kinase (MAPK) and stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK), but not p44/p42 MAPK. Anisomycin caused EGFR degradation and this was abolished by a specific p38 MAPK inhibitor, SB203580. Surprisingly, whereas EGF induced phosphorylation at Tyr1045, but not Ser1046/7, anisomycin induced the phosphorylation of EGFR at Ser1046/7, but not Tyr1045. In addition, though both EGF and anisomycin caused EGFR internalization, the EGFR internalized by anisomycin was not associated with an ubiquitin ligase, c-Cbl. Furthermore, SB203580 or gene silencing using p38 MAPK-siRNA suppressed anisomycin-induced phosphorylation of EGFR at Ser1046/7. These results strongly suggest that p38 MAPK directs EGFR toward desensitization via its phosphorylation at Ser1046/7.     

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.     

The inhibition of ERK/MAPK not the activation of JNK/SAPK is primarily required to induce apoptosis in chronic myelogenous leukemic K562 cells

In this study, the downstream signaling of Bcr-Abl tyrosine kinase responsible for apoptosis resistance was investigated. DNA fragmentation, a hallmark of apoptosis, was observed after 2 days of herbimycin A treatment with a peak on 3 day. During the apoptosis induced by the treatment of herbimycin A, stress-activated protein kinase (SAPK) and p38 kinase were activated time- and dose-dependently, while extracellular signal-regulated kinase (ERK) was inhibited. However, apoptosis was induced by the treatment of PD98059, a specific inhibitor of MEK (MAPK or ERK kinase), not by the treatment of sorbitol, a strong activator of SAPK and p38 kinase. Although K562 cells were very resistant to sorbitol-induced apoptosis, DNA fragmentation was induced rapidly in Jurkat, HL-60 and U937 cells after exposure to sorbitol, despite that these apoptosis-sensitive cells have similar or lower activities of JNK/SAPK and p38 kinase compared with K562 cells after treatment of sorbitol. K562 cells had a much higher basal activity of ERK/MAPK than other apoptosis-sensitive cell lines, which were very susceptible to apoptosis induced by low dose of PD98059 compared with K562 cells. In HL-60 cells, sorbitol-induced apoptosis was prevented by the treatment of phorbol myristate 13-acetate (PMA), which activates the ERK/MAPK pathway, and this was blocked by PD98059. From these results, it could be suggested that the inhibition of ERK/MAPK not the activation of JNK/SAPK is primarily required to induce apoptosis in K562 cells.     

Multiple signal pathways are involved in the mitogenic effect of 5(S)-HETE in human pancreatic cancer

Pancreatic carcinoma is characterized by poor prognosis and lack of response to conventional therapy. The reasons for this are not fully understood. We have reported that inhibition of 5-lipoxygenase abolished proliferation and induced apoptosis in pancreatic cancer cells while the 5-lipoxygenase metabolite, 5(S)-hydroxyeicosatetraenoic acid [5(S)-HETE] stimulated pancreatic cancer cell proliferation. The current study was designed to investigate the underlying mechanisms for 5(S)-HETE-stimulated proliferation of pancreatic cells. Two human pancreatic cancer cell lines, PANC-1 and HPAF, were used. Cell proliferation was monitored by thymidine incorporation and cell counting. Phosphorylation of P42/44(MAPK) (mitogen activated protein kinase, ERK), MEK (MAPK/ERK kinase), P38 kinase, JNK/SAPK (c-Jun N-terminal kinase/ stress-activated protein kinase), AKT and tyrosine residues of intracellular proteins was measured by Western blot using their corresponding phospho-specific antibodies. The results showed that (1) 5(S)-HETE markedly stimulated pancreatic cancer cell proliferation in a time- and concentration-dependent manner; (2) 5(S)-HETE induced tyrosine phosphorylation of multiple intracellular proteins while the tyrosine kinase inhibitor, genestein, blocked 5(S)-HETE-stimulated cell proliferation; (3) 5(S)-HETE significantly stimulated both MEK and P42/44(MAPK) phosphorylation and the MEK inhibitors, PD098059 and U0126, inhibited 5(S)-HETE-stimulated proliferation in these two cell lines; (4) 5(S)-HETE also stimulated P38 kinase phosphorylation but the P38 inhibitor, SB203580, did not effect 5(S)-HETE-stimulated cell proliferation; (5) 5(S)-HETE markedly stimulated AKT phosphorylation while the phosphatidylinositide-3 (PI3)-kinase inhibitor, wortmannin, blocked 5(S)-HETE-stimulated cell proliferation; (6) phosphorylation of JNK/SAPK was not induced by 5(S)-HETE, and (7) the general protein kinase C (PKC) inhibitor, GF109203X, did not affect 5(S)-HETE-stimulated cancer cell proliferation. These findings suggest that intracellular tyrosine kinases, MEK/ERK and PI3 kinase/AKT pathways are involved in 5(S)-HETE-stimulated pancreatic cancer cell proliferation but P38 kinase, JNK/SAPK and PKC are not involved in this mitogenic effect.     

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.     

Src-dependent ERK5 and Src/EGFR-dependent ERK1/2 activation is required for cell proliferation by asbestos

Crocidolite asbestos elicits oxidative stress and cell proliferation, but the signaling cascades linked to these outcomes are unclear. To determine the role of mitogen-activated protein kinases (MAPK) in asbestos-induced cell signaling, we evaluated the effects of crocidolite asbestos, EGF and H2O2, on MAPK activation in murine lung epithelial cells (C10 line). In contrast to rapid and transient activation of extracellular signal-regulated kinase 5 (ERK5) by EGF or H2O2, asbestos caused protracted oxidant-dependent ERK5 activation that was inhibited by an Src kinase inhibitor (PP2), but not by an inhibitor of epidermal growth factor receptor (EGFR) phosphorylation (AG1478). ERK1/2 activation by asbestos was inhibited by either PP2 or AG1478. To confirm the involvement of Src in ERK1/2 and ERK5 activation, a dominant-negative Src construct was used. These experiments showed that Src was essential for ERK1/2 and also ERK5 phosphorylation by asbestos. Time frame studies indicated immediate activation of Src by asbestos fibers, whereas EGFR phosphorylation occurred subsequently. Data suggest that asbestos causes activation of ERK5 through an EGFR-independent pathway, whereas ERK1/2 activation is dependent on Src through a mechanism involving phosphorylation of the EGFR. Furthermore, Src, ERK1/2 and ERK5 activation are essential for cell proliferation by asbestos. The use of a dominant-negative ERK5 construct caused selective downregulation of c-jun expression, whereas inhibition of Src by PP2 or MEK1 by PD98059 caused decreases in c-fos, fra-1 and c-jun expression in asbestos-exposed C10 cells. These observations may have broad relevance to cell proliferation by carcinogenic mineral fibers and oxidants.     

Mitogen-activated protein kinase pathway is dispensable for microtubule-active drug-induced Raf-1/Bcl-2 phosphorylation and apoptosis in leukemia cells.

Raf-1 activation and Bcl-2 hyperphosphorylation following treatment with paclitaxel (Taxol) or other microtubule-active drugs is associated with mitotic arrest. Here we show that microtubule-active drugs do not activate the mitogen-activated protein kinase (MAPK) pathway in leukemia cells. PD98059, a MEK inhibitor, and SB202190, a p38 MAP kinase inhibitor, do not abrogate Bcl-2 phosphorylation nor apoptosis. Simultaneously with PARP cleavage, paclitaxel induces cleavage of Bcl-2 protein yielding a potentially pro-apoptotic 22 kDa product. In comparison, the stimulation of Raf-1 by phorbol ester (TPA) activates the MAPK pathway, causes MAPK-dependent p21WAF1/CIP1 induction, Rb dephosphorylation and growth arrest without Bcl-2 phosphorylation or apoptosis. Like TPA, cAMP induces p21WAF1/CIP1 but does not cause Bcl-2 phosphorylation. MEKK1 and Ras, upstream activators of JNK and ERK MAPK, also fail to induce Bcl-2 hyperphosphorylation. Although Lck tyrosine kinase has been recently implicated in Raf-1 activation during mitotic arrest, microtubule-active drugs induce Raf-1/Bcl-2 hyperphosphorylation and apoptosis in a Lck-deficient Jurkat cells. Therefore, microtubule-active drugs induce apoptosis which is associated with Raf-1 and Bcl-2 phosphorylation and Bcl-2 cleavage but is independent of the MAPK pathway. In contrast, TPA-activated MAPK pathway causes p21WAF1/CIP1-dependent growth arrest without apoptosis.     

Activation of RSK by UV-light: phosphorylation dynamics and involvement of the MAPK pathway

Ribosomal S6 kinases (RSKs) are serine/threonine kinases activated by mitogenic signals through the Mitogen-Activated Protein Kinases/Extracellular Signal-Regulated Kinases (MAPK/ERK). RSKs contain two heterologous complete protein kinase domains. Phosphorylation by ERK of the C-terminal kinase domain allows activation of the N-terminal kinase domain, which mediates substrate phosphorylation. In human, there are three isoforms of RSK (RSK1, RSK2, RSK3), whose functional specificity remains undefined. Importantly, we have shown that mutations in the RSK2 gene lead to the Coffin-Lowry syndrome (CLS). In this study, we characterize two monoclonal antibodies raised against phosphorylated forms of the N- and C-terminal domain of RSK2 (P-S227 and P-T577, respectively). Using these two antibodies, we show that stress signals, such as UV light, induce phosphorylation and activation of the three RSKs to an extent which is comparable to Epidermal Growth Factor (EGF)-mediated activation. The use of specific kinase inhibitors indicates that UV-induced phosphorylation and activation of RSK2 is mediated by the MAPK/ERK pathway, but that the Stress-Activated Protein Kinase 2 (SAPK2)/p38 pathway is also involved. These results modify the view of RSKs as kinases restricted to the mitogenic response and reveal a previously unappreciated role of MAPKs in stress induced signaling. Oncogene (2000) 19, 4221 - 4229     

Role of mitogen-activated protein kinase phosphatases (MKPs) in cancer

The mitogen-activated protein kinase (MAPK) phosphatases (MKPs) are a family of dual-specificity protein phosphatases that dephosphorylate both phospho-threonine and phospho-tyrosine residues in MAP kinases, including the c-Jun N-terminal protein kinase (JNK)/stress-activated protein kinase (SAPK), the p38 MAPK, and the extracellular signal-related kinase (ERK). Since phosphorylation is required for the activation of MAP kinases, dephosphorylation by MKPs inhibits MAPK activity, thereby negatively regulating MAPK signaling. It is known that deregulation of MAPK signaling is the most common alteration in human cancers. Recent studies have suggested that MKPs play an important role not only in the development of cancers, but also in the response of cancer cells to chemotherapy. Thus, understanding the roles of MKPs in the development of cancer and their impact on chemotherapy can be exploited for therapeutic benefits for the treatment of human cancer. This paper received support from NIH grant R01 CA100073 and Elsa U. Pardee Foundation.