RNA interference (RNAi) for extracellular signal-regulated kinase 1 (ERK1) alone is sufficient to suppress cell viability in ovarian cancer cells

While ovarian cancer is a leading cause of death in females today, the molecular, genetic, and environmental factors that initiate and support the progression of this disease are still only partially understood. The extracellular signal-regulated kinase (ERK) signaling pathway is a major contributor to cellular growth, differentiation and survival. Recently, we reported that this pathway is constitutively activated in ovarian cancer cells, and that by using RNA interference (RNAi) for ERK1 and ERK2, we were able to significantly suppress the number of viable tumor cells. In the present study, we have further investigated the mechanisms by which RNAi for the ERK kinases decreased viability in these cancer cells. It was determined that treatment of the cancer cells with small inhibitory RNAs (siRNAs) directed against ERK1 and ERK2 leads to the induction of apoptosis and necrosis by four hours following treatment. Additionally, we found that primary, nonmalignant ovarian cells do not respond similarly to ERK siRNA treatment and that these cells fail to die following treatment. Data presented show that ERK2 expression is more difficult to silence, depending upon cell type being examined and that silencing ERK1 expression alone is sufficient to significantly decrease tumor cell viability.     

Fas drives cell cycle progression in glioma cells via extracellular signal-regulated kinase activation

Recent studies have revealed that a variety of malignant tumors express Fas and/or its ligand FasL. However, tumor cells expressing Fas are not always susceptible to Fas-mediated cell death, and the biological significance of simultaneous expression of Fas and FasL in the same tumor is not known. In the present study, we addressed this question in three glioma cells lines, A-172, T98G, and YKG-1, which express both Fas and FasL endogenously and their Fas transfectants. We report here that: (a) in gliomas, [3H]TdR incorporation was enhanced by anti-Fas IgM monoclonal antibody CH-11 and conversely inhibited by anti-FasL monoclonal antibody NOK-2; (b) cross-linking of Fas with CH-11 drove both cell cycle progression and apoptosis as demonstrated by the induction of the S-G2 phase of DNA and RNA and fragmented nuclei; (c) phosphorylation of extracellular signal-regulated kinase (ERK), but not of c-Jun NH2-terminal kinase or p38, was induced by cross-linking of Fas; (d) a mitogen-activated protein kinase/ERK kinase 1 (MEK1) inhibitor PD98059 completely blocked CH-11-induced ERK phosphorylation as well as cell cycle progression without affecting induction of apoptosis; and (e) a broad-spectrum caspase inhibitor Z-Asp-CH2-DCB inhibited CH-11-induced ERK phosphorylation, cell cycle progression, and apoptosis. These results indicate that Fas-mediated caspase activation elicits two independent cellular responses; one is to induce apoptosis and another is to promote cell cycle progression; the latter is closely linked to the MEK-ERK pathway. Together, our data strongly suggest that FasL may play a role as an autocrine growth factor in gliomas.     

Activation of the RAF/mitogen-activated protein/extracellular signal-regulated kinase kinase/extracellular signal-regulated kinase pathway mediates apoptosis induced by chelerythrine in osteosarcoma

PURPOSE: Chelerythrine, a widely used broad-range protein kinase C inhibitor, induces apoptosis in many cell types. In this study, the mechanism of chelerythrine-induced apoptosis in osteosarcoma was investigated. EXPERIMENTAL DESIGN: Signaling pathways activated by chelerythrine in osteosarcoma were detected by Western blots. Impacts of RAF/mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) kinase (MEK)/ERK MAPK on apoptosis and cell survival were studied using genetic approaches and pharmacologic pathway-specific inhibitors. RESULTS: Osteosarcoma cells underwent apoptosis rapidly after treatment with chelerythrine. Three parallel MAPKs pathways, including the ERKs, c-Jun NH(2) kinases, and p38, were activated by chelerythrine in a dose-dependent and time-dependent fashion. For the ERKs, the activation was evident at the earliest time point tested (2 minutes) and sustained for >4 hours. Introduction of a dominant-negative H-RAS mutant (17N) partially attenuated ERK activation and delayed the onset of apoptosis induced by chelerythrine. The ERK activation and apoptotic effects of chelerythrine were greatly abrogated by the pharmaceutical inhibitors of MEK, but not by those of c-Jun NH(2) kinase or p38. Moreover, osteosarcoma cells were sensitized to chelerythrine by transient transfection with wild-type MEK1 or constitutively active MEK1 and became resistant with dominant-negative MEK1. Other protein kinase C inhibitors, including GF109203X or G?6976, did not cause ERK activation or apoptosis in the same timeframe tested. CONCLUSION: In osteosarcoma, chelerythrine-induced apoptosis is mediated through activation of the RAF/MEK/ERK pathway. These findings suggest that activating the ERK MAPK, as opposed to inhibiting it, may be a therapeutic strategy in osteosarcoma.     

细胞外信号调节激酶在直肠癌发生发展中的意义

Objective： To study the ERK expression and its significance in the development of human rectal carcinoma. Methods： Samples were obtained from 62 patients with rectal carcinoma, including tumor tissue and adjacent normal rectal tissue. Western blot was used to measure the expression of ERK-1 and ERK-2. S-P immunohistochemical method was used to count the microvessel density （MVD）. Results： ERK-1 and ERK-2 protein levels were increased in rectal carcinoma tissue compared with those in adjacent normal tissues （t = 2.980 and 2.194, P 〈 0.01 and 0.05 respectively）. The MVD was higher in patients with higher ERK-1 and/or ERK-2 protein levels than that in cases with lower ERK-1 and/or ERK-2 levels. Conclusion： Overexpression of ERK-1 and/or ERK-2 may play an important role in the development of human rectal carcinoma, the overexpression can enhance the growth of tumor microvessel and promote the development of human rectal carcinoma.     

Selective reduction of extracellular signal-regulated protein kinase (ERK) phosphorylation in squamous cell carcinoma of the larynx

Mitogen-activated protein kinase (MAPK) cascades transmit and amplify signals involved in cell proliferation as well as in cell death. In this study, the potential derangement of MAPK pathways has been evaluated in human squamous cell carcinomas (SCC) of the larynx. The expression and activity of the MAPK p38, ERK1/2p44/p42 and JNK/SAPKp46/p54 have been investigated by immunoblot analysis of tissue homogenates in 27 samples of primary laryngeal cancer and in 27 paired non-neoplastic laryngeal mucosa. On the same tissues, the activation of MAPK JNK/SAPKp46/p54 was also analyzed by an ELISA assay. The results obtained showed that both total and phosphorylated levels of JNK/SAPKp46/p54 and p38 were not different between tumor and normal samples. Conversely, while total protein levels for both ERK1p44 and ERK2p42 were not statistically different between tumor and normal samples, the analysis of the level of the activated forms of ERK1/2 showed a statistically significant decreased phosphorylation of both isoforms in the tumor samples compared to the control tissues. The rate of reduction was similar for both isoforms. Immunohistochemical analysis of all the activated MAPK (p38, JNK/SAPKp46/p54 and ERK1/2p44/p42) in both laryngeal SCC and normal mucosa demonstrated no difference of cellular localization. Activated ERK1/2p44/p42 and activated p38 demonstrated a nucleo-cytoplasmic distribution whereas activated JNK/SAPKp46/p54 were localized into the cytoplasmic membrane. The decreased activity of ERK1/2p44/42 in laryngeal SCC might reflect alterations in tumor suppressing activity or might derive from the interplay among various transduction pathways.     

Critical role of extracellular signal-regulated kinase (ERK) phosphorylation in novel vitamin K analog-induced cell death.

In the present study, we show that 2-(2-hydroxyethylsulfaryl)-3-methyl-1,4-naphthoquinone, or CPD 5, is a potent growth inhibitor for pancreas cancer cell lines (ID(50): 21.4 +/- 3.8, 31.8 +/- 2.7 and 55.2 +/- 4.5 microM for MiaPaCa, Panc-1 and BxPc3, respectively). It induced protein tyrosine phosphor-ylation of hepatocyte growth factor (HGF) receptor (c-Met) or epidermal growth factor receptor (EGFR), which increased progressively to a maximum level at 30 min in Panc-1 cells. The receptor phosphorylation by CPD 5 was indicated to be functional, since these receptors were found to bind with Grb2 or SOS1 protein. CPD 5 was also suggested to induce phosphorylation of external signal-regulated kinase (ERK). EGF induced cell proliferation through ERK phosphorylation, since U0126, which is an inhibitor of ERK phosphorylation, abrogated the increase of cyclin D1 by EGF. HGF increased the amount of p27 protein, suggesting that it is associated with cell differentiation. By contrast, U0126 reduced CPD 5-induced cell death. On two-dimensional electrophoresis, we found an extra type of phospho-ERK, and this was completely and selectively abolished by U0126. These results suggest that ERK phosphorylation, especially the extra spot on two-dimensional gel, is critically associated with CPD 5-mediated cell death.     

Inhibition of extracellular signal-regulated kinase 1/2 phosphorylation and induction of apoptosis by sulindac metabolites

Regular use of nonsteroidal anti-inflammatory drugs (NSAIDs) such as aspirin and sulindac is associated with a decreased mortality from colorectal cancer. Sulindac causes regression of precancerous adenomatous polyps and inhibits the growth of cultured colon cell lines. Whereas induction of apoptotic cell death is thought to account for the growth inhibitory effect of sulindac, less is known about its biochemical mechanism(s) of action. Sulindac is metabolized in vivo to sulfide and sulfone derivatives. Both the sulfide and sulfone metabolites of sulindac as well as more potent cyclic GMP-dependent phosphodiesterase inhibitors were shown to cause inhibition of extracellular signal-regulated kinase (ERK)1/2 phosphorylation at doses (40-600 microM) and times (1-5 days) consistent with the induction of apoptosis by the drugs. Treatment of HCT116 human colon cancer cells with the specific mitogen-activated protein kinase kinase, U0126 (5-50 microM) resulted in a time- and dose-dependent inhibition of ERK1/2 phosphorylation, and induction of apoptosis. U0126 treatment (20 microM) increased basal apoptosis, and potentiated the apoptotic effect of sulindac sulfide and sulindac sulfone. These results suggest that the inhibition of ERK1/2 phosphorylation is responsible for at least part of the induction of programmed cell death by sulindac metabolites. Inhibition of ERK1/2 activity may, therefore, be a useful biochemical target for the development of chemopreventive and chemotherapeutic drugs for human colon cancer.     

Role of nongenomic activation of phosphatidylinositol 3-kinase/Akt and mitogen-activated protein kinase/extracellular signal-regulated kinase kinase/extracellular signal-regulated kinase 1/2 pathways in 1,25D3-mediated apoptosis in squamous cell carcinoma cells

Vitamin D is a steroid hormone that regulates calcium homeostasis and bone metabolism. The active form of vitamin D [1 alpha,25-dihydroxyvitamin D(3) (1,25D3)] acts through both genomic and nongenomic pathways. 1,25D3 has antitumor effects in a variety of cancers, including colorectal, prostate, breast, ovarian, and skin cancers. 1,25D3 exerts growth-inhibitory effects in cancer cells through the induction of apoptosis, cell cycle arrest, and differentiation. The mechanisms regulating 1,25D3-induced apoptosis remain unclear. We investigated the role of nongenomic signaling in 1,25D3-mediated apoptosis in squamous cell carcinoma (SCC) cells. 1,25D3 induced rapid and sustained activation of phosphatidylinositol 3-kinase/Akt and mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK) 1/2 pathways in SCC cells. These effects were nongenomic: they occurred rapidly and were not inhibited by cycloheximide or actinomycin D. To examine whether the nongenomic activation of Akt and ERK1/2 plays a role in 1,25D3-mediated apoptosis, the expression of Akt or ERK1/2 was reduced by small interfering RNA (siRNA). siRNA-Akt significantly enhanced 1,25D3-induced apoptosis as indicated by increased levels of Annexin V-positive cells and increased sub-G(1) population and DNA fragmentation. In contrast, siRNA-ERK1/2 had no effects on 1,25D3-induced apoptosis. In addition, siRNA-Akt transfection followed by 1,25D3 treatment induced apoptosis much sooner than 1,25D3 alone. siRNA-Akt and 1,25D3 induced caspase-10 activation, suppressed the expression of c-IAP1 and XIAP, and promoted 1,25D3-induced caspase-3 activation. These results support a link between 1,25D3-induced nongenomic signaling and apoptosis. 1,25D3 induces the activation of phosphatidylinositol 3-kinase/Akt, which suppresses 1,25D3-mediated apoptosis and prolongs the survival of SCC cells.     

The duration of nuclear extracellular signal-regulated kinase 1 and 2 signaling during cell cycle reentry distinguishes proliferation from apoptosis in response to asbestos

Asbestos exposure causes activation of extracellular signal-regulated kinases 1 and 2 (ERK1/2) in lung epithelial cells, the targets of asbestos-associated lung carcinomas. The functional significance of ERK1/2 activation in pulmonary epithelial and mesothelial cells is unclear. Using serum-stimulated mouse alveolar type II epithelial cells as a model for cell cycle reentry, we show that the duration of phospho-ERK1/2 in the nucleus determines cell fate in response to crocidolite asbestos. In response to 10% serum, a proliferative stimulus, phosphorylated ERK1/2 initially accumulated in the nucleus, and reduction of nuclear phospho-ERK1/2 after 2 to 4 hours was followed by expression of cyclin D1 and S-phase entry. Low levels of asbestos (<0.5 microg/cm2) promoted S-phase entry in low (2%) serum through an epidermal growth factor receptor-dependent pathway but did not promote cell cycle progression or induce apoptosis in the presence of high (10%) serum-containing medium. Higher levels of asbestos (1.0 to 5.0 microg/cm2) prolonged the localization of phospho-ERK1/2 in the nucleus in the presence of high serum, impeded S-phase entry, and induced apoptosis in a dose-dependent manner. Immunofluorescence microscopy indicated that the duration of signaling by phospho-ERK1/2 in the nucleus was predictive of cell fate at any concentration of asbestos. After 8 hours of exposure, cells with nuclear phospho-ERK1/2 also were positive for nuclear localization of apoptosis-inducing factor (AIF), an early event in apoptosis. In contrast, asbestos-exposed cells that displayed cytoplasmic phospho-ERK1/2 at 8 hours expressed cyclin D1 and proceeded to S phase. Our studies show that prolonged localization of phospho-ERK1/2 in the nucleus is incompatible with expression of cyclin D1 and is predictive of asbestos-associated cell death by AIF, thereby providing an approach for determining cell fate in asbestos-induced tumorigenesis.     

细胞外信号调节激酶1/2参与三氧化二砷诱导的胶质瘤细胞凋亡

目的:探讨细胞外信号调节激酶1/2(ERK1/2)信号通路在三氧化二砷诱导的胶质瘤细胞U251凋亡中的作用,为应用三氧化二砷治疗胶质瘤奠定基础。方法:50μmol/L三氧化二砷作用U251细胞,不同时间检测胶质瘤细胞增殖活性,半定量PCR检测ERK1/2mRNA表达,Western blot检测ERK1/2蛋白表达;Ho-echst33258染色及流式细胞术(FCM)检测细胞凋亡;转染ERK1/2上游激酶MEK1,应用ERK1/2激酶抑制剂U0126观察ERK1/2通路在肿瘤凋亡及增殖中的作用;比色分析法检测Caspase-3活性的变化。结果:三氧化二砷诱导胶质瘤细胞发生明显凋亡,抑制肿瘤细胞增殖;增加ERK1/2蛋白的表达,呈时间依赖性;阻断ERK1/2信号通路后胶质瘤细胞凋亡受到抑制,Caspase-3活性下降。结论:ERK1/2信号通路在三氧化二砷诱导的胶质瘤细胞凋亡中起重要作用。     

Heregulin beta1-activated phosphatidylinositol 3-kinase enhances aggregation of MCF-7 breast cancer cells independent of extracellular signal-regulated kinase

Heregulin (HRG) is a family of polypeptide growth factors derived from alternatively spliced genes. HRG can bind to receptor tyrosine kinases erbB3 and erbB4, thereby inducing erbB3 and erbB4 heterodimerization with erbB2, leading to receptor tyrosine phosphorylation and activating downstream signal transduction. Cell-cell homophilic adhesion (cell aggregation) is important in determining the structural organization and behavior of cells in tissues. In addition, tumor cell homophilic adhesion may affect invasive and metastatic potentials of cells. We report that HRG-beta1 can enhance aggregation of MCF-7 and SKBR3 human breast cancer cells. While investigating the downstream signals involved in HRG-beta1-enhanced cell aggregation, we observed that HRG-beta1 induced tyrosine phosphorylation of erbB2 and crbB3 receptor heterodimers and increased the association of the dimerized receptors with the 85-kDa subunit of phosphatidylinositol 3-kinase (PI3K). HRG-beta also increased the kinase activities of extracellular signal-regulated protein kinase (ERK) and PI3K in these cells. By using the mitogen-activated protein kinase/ERK 1 (MEK1) inhibitor PD98059 and PI3K inhibitors wortmannin and LY294002, we found that blocking the MEK1-ERK pathway had no effect on HRG-pbeta1-enhanced cell aggregation; however, blocking the PI3K pathway greatly inhibited HRG-beta1-mediated cell aggregation. Our study indicated that the HRG-beta1-activated MEK1-ERK pathway has no demonstrable role in the induction of cell aggregation, whereas HRG-beta1-activated PI3K is required for enhancing breast cancer cell aggregation. Because aggregation can contribute to invasion/metastasis phenotype of cancer cells, our results have provided one mechanism by which HRG-beta1-activated signaling of erbB receptors may affect invasive/metastatic properties of MCF-7 and SKBR3 breast cancer cells.     

The kinase-inhibitory domain of p21-activated kinase 1 (PAK1) inhibits cell cycle progression independent of PAK1 kinase activity

p21-activated kinase 1 (PAK1) is a mediator of downstream signaling from the small GTPases Rac and Cdc42. In its inactive state, PAK1 forms a homodimer where two kinases inhibit each other in trans. The kinase inhibitory domain (KID) of one molecule of PAK1 binds to the kinase domain of its counterpart and keeps it inactive. Therefore, the isolated KID of PAK1 has been widely used to specifically inhibit and study PAK function. Here, we show that the isolated KID induced a cell cycle arrest with accumulation of cells in the G1 phase of the cell cycle with an inhibition of cyclin D1 and D2 expression. This cell cycle arrest required the intact KID and was also induced by a mutated KID unable to block PAK1 kinase activity. Furthermore, the KID-induced cell cycle arrest could not be rescued by the expression of a constitutively active PAK1-T423E mutant, concluding that this arrest occurs independently of PAK1 kinase activity. Our results suggest that PAK1 through its KID inhibits cyclin D expression and thereby enforces a cell cycle arrest. Our results also call for serious precaution in the use of KID to study PAK function.     

p44 mitogen-activated protein kinase (extracellular signal-regulated kinase 1)-dependent signaling contributes to epithelial skin carcinogenesis

Extracellular signal-regulated kinases (ERK) regulate cellular functions in response to a variety of external signals. However, the specific functions of individual ERK isoforms are largely unknown. Hence, we have investigated the specific function of ERK1 in skin homeostasis and tumorigenesis in ERK1 knockout mice. They spontaneously develop cutaneous lesions and hyperkeratosis with epidermis thickness. Skin hyperproliferation and inflammation induced by application of 12-O-tetradecanoylphorbol-13-acetate (TPA) is strongly reduced in mutant mice. ERK1(-/-) mice are resistant to development of skin papillomas induced by 7,12-dimethylbenz(a)anthracene (DMBA) and promoted by TPA. Tumor appearance was delayed, their formation was less frequent, and their number and size were reduced. Keratinocytes obtained from knockout mice showed reduced growth and resistance to apoptotic signals, accompanied by an impaired expression of genes implicated in growth control and invasiveness. These results highlight the importance of ERK1 in skin homeostasis and in the process of skin tumor development.     

Annexin V inhibits the 12-O-tetradecanoylphorbol-13-acetate-induced activation of Ras/extracellular signal-regulated kinase (ERK) signaling pathway upstream of Shc in MCF-7 cells

Annexin V is a Ca2+-dependent phospholipid binding protein. Although it has been shown to inhibit protein kinase C (PKC) in cell-free systems, its role in the intact cell is unclear. A stable MCF-7 human breast cancer cell overexpression system was established to investigate the function of annexin V. In these cells, 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced phosphorylation and kinase activity of ERK1/2 were suppressed. Morphological changes induced by TPA were reduced by annexin V overexpression as well as by the pan-PKC inhibitor, bisindolylmaleimide I, and by the mitogen-activated protein kinase/extracellular signal-regulated kinase kinase (MEK) inhibitor, PD98059. TPA-induced MEK1/2 and Raf-1 phosphorylation were reduced in these cells. The TPA-enhanced active Ras, and its association with Raf-1, were reduced. TPA treatment of MCF-7 cells caused an increased association of Shc with Grb2. However, this increased association was prevented in the annexin V-overexpressors. p21WAF/CIP1 is responsible for inhibition of cell cycle progression in MCF-7 cells. TPA induced the expression of p21WAF/CIP1 to a greater extent in MCF-7 parent and control plasmid cells than in annexin V overexpressors. PD98059 inhibited this increase, suggesting that TPA upregulation of p21WAF/CIP1 occurs via the MEK pathway, and that annexin V overexpression blunts it. This work shows that annexin V overexpression suppresses the TPA-induced Ras/ERK signaling by inhibiting at/or upstream of Shc, possibly through the inhibition of PKCs. Oncogene (2000).     

Breast tumor kinase and extracellular signal-regulated kinase 5 mediate Met receptor signaling to cell migration in breast cancer cells

Introduction  

Breast tumor kinase (Brk/protein tyrosine kinase 6 (PTK6)) is a nonreceptor, soluble tyrosine kinase overexpressed in the majority of breast tumors. Previous work has placed Brk downstream of epidermal growth factor receptor (ErbB) activation and upstream of extracellular signal-regulated kinase 5 (ERK5) and p38 mitogen-activated protein (MAP) kinases. Herein we investigate the regulation of Brk kinase activity and cell migration in response to treatment of keratinocytes (HaCaT cells) and breast cancer cell lines (MDA-MB-231 and T47D cells) with hepatocyte growth factor (HGF) and macrophage stimulating protein (MSP), peptide ligands for Met and Ron receptors, respectively.