Targeting GRB7/ERK/FOXM1 Signaling Pathway Impairs Aggressiveness of Ovarian Cancer Cells

Ovarian cancer is a highly lethal disease with poor prognosis and especially in high-grade tumor. Emerging evidence has reported that aberrant upregulation and activation of GRB7, ERK as well as FOXM1 are closely associated with aggresivenesss of human cancers. However, the interplay between these factors in the pathogenesis of human cancers still remains unclear. In this study, we found that GRB7 (P<0.0001), ERK phosphorylation (P<0.0001) and FOXM1 (P = 0.001) were frequently increased and associated with high-grade tumors, as well as a high tendency in association with advanced stage ovarian cancer by immunohistochemical analysis. Intriguingly, the expressions of GRB7 (P<0.0001), ERK phosphorylation (P<0.001) and FOXM1 (P<0.001) showed a significant stepwise increase pattern along Grade 1 to Grade 3 ovarian cancers. Biochemical studies using western blot analysis demonstrated that enforced expression or knockdown of GRB7 showed GRB7 could elevate the levels of ERK phosphorylation and FOXM1, whereas enforced expression of FOXM1 could not alter levels of GRB7 and ERK phosphorylation. But inhibition of ERK signaling by U0126 or PD98059 could reduce the level of FOXM1 in GRB7-overexpressing ovarian cancer cells, suggesting that GRB7, ERK and FOXM1 are regulated orderly. Moreover, inhibition of ERK activity by U0126 or PD98059, or decreased FOXM1 expression by Thiostrepton significantly inhibited cell migration/invasion, tumor growth in vitro and in vivo. Collectively, our findings confer that targeting GRB7/ERK/FOXM1 signaling cascade may be a promising molecular therapeutic choice in combating ovarian cancer.     

A Non-canonical MEK/ERK Signaling Pathway Regulates Autophagy via Regulating Beclin 1

Autophagy-essential proteins are the molecular basis of protective or destructive autophagy machinery. However, little is known about the signaling mechanisms governing these proteins and the opposing consequences of autophagy in mammals. Here we report that a non-canonical MEK/ERK module, which is positioned downstream of AMP-activated protein kinase (AMPK) and upstream of tuberous sclerosis complex (TSC), regulates autophagy by regulating Beclin 1. Depletion of ERK partially inhibited autophagy, whereas specific inhibition on MEK completely inhibited autophagy. MEK could bypass ERK to promote autophagy. Basal MEK/ERK activity conferred basal Beclin 1 by preventing disassembly of mammalian target of rapamycin complex 1 (mTORC1) and mTORC2. Activation of MEK/ERK by AMPK upon autophagy stimuli disassembled mTORC1 via binding to and activating TSC but disassembled mTORC2 independently of TSC. Inhibition of mTORC1 or mTORC2 by transiently or moderately activated MEK/ERK caused moderately enhanced Beclin 1 resulting in cytoprotective autophagy, whereas inhibition of both mTORC1 and mTORC2 by sustained MEK/ERK activation caused strongly pronounced Beclin 1 leading to cytodestructive autophagy. Our findings thus propose that the AMPK-MEK/ERK-TSC-mTOR pathway regulation of Beclin 1 represents different thresholds responsible for a protective or destructive autophagy.Autophagy is an evolutionally conserved machinery involving the degradation and turnover of cytoplasmic material in lysosomes. Autophagy plays a role in cellular homeostasis (1), antiaging (2–4), development (1, 5), protection of the genome (6), and regulation of cell size (7). Autophagy may act as a means of defense against bacterium and virus invasion and be linked to various diseases including cancer (8–10), cardiomyopathy (11), and neurodegenerative disorders (12).Autophagy starts with the formation of an autophagosome, enclosed within a double membrane that engulfs part of the cytoplasm. During periods of autophagy stimuli, cells respond to either maintain the metabolism essential for survival or execute cell death. Autophagy-essential proteins (Atg)² are the molecular basis of autophagy machinery. About 30 Atg proteins in yeast and 10 in mammals have been identified. In yeast, the protein kinase target of rapamycin (TOR) mediates autophagy via Atg1-Atg13 kinase complex. Atg1 interacts with multiple components of the autophagic machinery through direct association, phosphorylation, and/or intracellular localization (13, 14).In mammalian systems, autophagosomes fuse with lysosomes to generate autophagolysosomes, which undergo a maturation process by fusing with endocytic compartments and lysosomes (15). Because it is not known how the Atg1 homolog acts in mammals, a different mechanism may be involved in regulating autophagy. Beclin 1/Atg6, microtubule-associated protein 1 light chain 3 (LC3)/Atg8, Atg5, Atg12, and Atg13 are essential for autophagosome formation in mammalian species (5, 16–20). Atg7 and Atg3 are required in the conjugation reaction between Atg12 and Atg5 and in the lipidation of LC3. During the formation of autophagosomes in mammalian cells, LC3 is lipidated via a ubiquitylation-like system (17, 21), generating a soluble form, LC3-I. LC3-I is further modified to a membrane-bound form, LC3-II, which is subsequently localized to autophagosomes and autolysosomes until being degraded by the lysosome.Beclin 1 was initially isolated as a B-cell lymphoma-2 (Bcl2)-interacting tumor suppressor in mammalian cells (22). Overexpression of Bcl2 attenuates the formation of the kinase complex Beclin 1-class III phosphatidylinositol 3-kinase (PI3KC3) essential for the formation of autophagosomes (23). The UV radiation resistance-associated gene tumor suppressor and the activating molecule in Beclin 1-regulated autophagy protein 1 (Ambra 1) were identified as new Beclin 1-binding partners that also regulate autophagy by regulating the Beclin 1-PI3KC3 kinase complex. Association of Beclin 1 with PI3KC3 is negatively regulated by Bcl2 (22) and positively regulated by UV radiation resistance-associated gene tumor suppressor and Ambra 1 (24, 25). Beclin 1 is homoallelically deleted in many human tumors. A decreased Beclin 1 level causes defective autophagy and breast cancer, but restoration of Beclin 1 induces autophagy and inhibits tumorigenicity of human breast cancer cells (18). These reports evidence the dependence on Beclin 1 for a functional autophagy mechanism.Diverse signaling pathways have been reported in the regulation of autophagy in mammalian cells (26, 27). In contrast to yeast, mammalian cells regulate autophagy via both class I and class III PI3K. Class I PI3K plays an inhibitory role, whereas class III PI3K kinase complex, which includes Beclin 1, plays a stimulatory role in autophagy by promoting the nucleation of autophagic vesicles (28, 29). A recent study also indicates that hVps15 is required in regulation of class III PI3K in mammalian cells (30). However, the signaling mechanisms controlling autophagy-essential proteins, in particular Beclin 1, and the opposing consequences of autophagy remain to be resolved.Our present studies identified and positioned a non-canonical MEK/ERK pathway downstream of AMPK and upstream of TSC and mTOR. This MEK/ERK module regulated autophagy via regulating the Beclin 1 level through the AMPK-MEK/ERK-TSC-mTOR pathway. Moderately enhanced Beclin 1 by transient or moderate activation of MEK/ERK and subsequent inhibition on mTORC1 and mTORC2 individually caused protective autophagy. Strongly pronounced Beclin 1 by sustained or strong activation of MEK/ERK followed by dual inhibition on mTORC1 and mTORC2 caused destructive autophagy. Our results thus reveal interesting Beclin 1 thresholds in regulating autophagy.     

MEK/ERK信号通路对人结膜上皮细胞增殖的影响及其机制研究

目的:探讨MEK/ERK信号通路对人结膜上皮细胞增殖的影响及其可能的机制。方法:采用不同浓度(0、12.5、25、50、100μmol/L)的MEK抑制剂PD98059处理人结膜上皮细胞(HConEpiC),通过CCK-8法检测不同浓度PD98059作用不同时间(0、12、24、48 h)对人结膜上皮细胞增殖的影响,Western blot检测不同浓度PD98059对人结膜上皮细胞ERK1/2、P-ERK1/2表达的影响。结果:相比对照组(0μmol/L),不同浓度(12.5、25、50、100μmol/L)PD98059处理后的人结膜上皮细胞增殖率明显下降,呈剂量-效应关系,且随处理时间增加(12、24、48 h)其抑制作用也显著增强,差异均有统计学意义(P0.05)。不同浓度PD98059处理人结膜上皮细胞24 h后,其ERK及p-ERK1/2表达随处理浓度增加而降低,与对照组(0μmol/L)相比差异有统计学意义(P0.05),且二者表达量与细胞增值抑制率均呈显著负相关(r=-0.995、r=-0.968,P0.05)。结论:PD98059可抑制人结膜上皮细胞增殖,这可能与其下调ERK表达和减少其活化有关。     

Targeting Cadherin-17 Inactivates Ras/Raf/MEK/ERK Signaling and Inhibits Cell Proliferation in Gastric Cancer

Cadherin-17 (CDH17), one member of 7D-cadherin superfamily, was overexpressed in gastric cancer (GC) and was associated with poor survival, tumor recurrence, metastasis, and advanced tumor stage. So far the cellular function and signaling mechanism of CDH17 in GC remains unclear. In this study, we showed that over 66% of GC cell lines (20/30) were CDH17 positive. Tissue microarray (TMA) assay showed that 73.6% Chinese GC tissues (159/216) were CDH17 positive, while 37% respective adjacent normal tissues were CDH17 positive. Knockdown of CDH17 inhibited cell proliferation, migration, adhesion and colony formation, and also induced a cell cycle arrest and apoptosis in AGS human GC cells. On the other side, overexpression of CDH17 facilitated MGC-803 GC tumor growth in nude mice. Antibody array and Western blotting assay demonstrated that knockdown of CDH17 in AGS cells down-regulated integrin β series proteins, further inactivated the Ras/Raf/MEK/ERK pathway and led to p53 and p21 accumulation, which resulted in proliferation inhibition, cell-cycle arrest and apoptosis induction. Collectively, our data firstly demonstrate the capacity of CDH17 to regulate the activity of Ras/Raf/MEK/ERK pathway for cell proliferation in GC, and suggest that CDH17 can serve as an attractive therapeutic target for future research.     

MEK/ERK信号通路与脑缺血再灌注损伤

在脑缺血病灶中,中心区神经元坏死为主,周围以缺血半暗带凋亡为主,抑制半暗带细胞的凋亡,可以减少细胞的死亡和脑梗死的面积,因此改善半暗带是治疗脑卒中的关键环节.目前发现MAPK分布于整个中枢神经系统中,MEK/ERK信号通路参与细胞生长、发育、细胞抗凋亡等过程,在脑缺血再灌注损伤过程中有MEK/ERK信号通路的参与,MEK/ERK通路通过影响Bcl-2家族成员的活化和表达调控内源性凋亡途径,ERK通过对细胞周期的调控,抑制胶质细胞大量活化和过度增殖,减少了有害因子并改善局部微循环,从而减少神经元的凋亡.可能为脑血管的防治开辟一条新的途径.本文就MEK/ERK信号通路的结构特点与脑缺血再灌注损伤相关作用机制作一综述.     

Regulatory T Cells Negatively Affect IL-2 Production of Effector T Cells through CD39/Adenosine Pathway in HIV Infection

The mechanisms by which Regulatory T cells suppress IL-2 production of effector CD4+ T cells in pathological conditions are unclear. A subpopulation of human Treg expresses the ectoenzyme CD39, which in association with CD73 converts ATP/ADP/AMP to adenosine. We show here that Treg/CD39+ suppress IL-2 expression of activated CD4+ T-cells more efficiently than Treg/CD39−. This inhibition is due to the demethylation of an essential CpG site of the il-2 gene promoter, which was reversed by an anti-CD39 mAb. By recapitulating the events downstream CD39/adenosine receptor (A2AR) axis, we show that A2AR agonist and soluble cAMP inhibit CpG site demethylation of the il-2 gene promoter. A high frequency of Treg/CD39+ is associated with a low clinical outcome in HIV infection. We show here that CD4+ T-cells from HIV-1 infected individuals express high levels of A2AR and intracellular cAMP. Following in vitro stimulation, these cells exhibit a lower degree of demethylation of il-2 gene promoter associated with a lower expression of IL-2, compared to healthy individuals. These results extend previous data on the role of Treg in HIV infection by filling the gap between expansion of Treg/CD39+ in HIV infection and the suppression of CD4+ T-cell function through inhibition of IL-2 production.     

Cardiac hypertrophy: Targeting Raf/MEK/ERK1/2-signaling

Over the past two decades, basic research has revealed a complex network of regulatory mechanisms that control the ERK1/2-signaling cascade. ERK1/2 mediate cardiac hypertrophy, a major risk factor for the development of arrhythmias, heart failure and sudden death, but also beneficial effects, e.g. protection of the heart from cell death and ischemic injury. Selective targeting of these ambiguous ERK functions could provide a powerful tool in the treatment of cardiac disease. This short review will discuss new mechanistic insights into ERK1/2-dependent development of cardiac hypertrophy and the prospect to translate this knowledge into future therapeutic strategies.     

Homeostasis and Function of Regulatory T Cells in HIV/SIV Infection

Regulatory T cells (Tregs) play a pivotal role in the maintenance of tolerance as well as in the control of immune activation, particularly during chronic infections. In the setting of HIV infection, the majority of studies have reported an increase in Treg frequency but a decrease in absolute number in all immune compartments of HIV-infected individuals. Several nonexclusive mechanisms have been postulated to explain this preferential Treg accumulation, including peripheral survival, increased proliferation, increased peripheral conversion, and tissue redistribution. The role played by Tregs during HIV infection is still poorly understood, as two opposing hypotheses have been proposed. A detrimental role of Tregs during HIV infection was suggested based on the evidence that Tregs suppress virus-specific immune responses. Conversely, Tregs could be beneficial by limiting immune activation, thus controlling the availability of HIV targets as well as preventing immune-based pathologies. Despite the technical difficulties, getting a better understanding of the mechanisms regulating Treg dynamics remains important, as it will help determine whether we can successfully manipulate Treg function or number to the advantage of the infected host. The aim of this review is thus to discuss the recent findings on Treg homeostasis and function in the setting of HIV infection.     

ERK1/2信号通路活化对Tamoxifen所致胶质瘤细胞凋亡的影响

为了探讨ERK1/2信号通路在他莫昔芬(tamoxifen, TAM)所致胶质瘤细胞凋亡中的作用,以C6和U87MG胶质瘤细胞为研究对象,经TAM处理后,采用MTT法检测细胞的存活率;倒置显微镜和DAPI染色观察细胞的形态;流式细胞术检测细胞凋亡; Western-blot法检测细胞内ERK1/2磷酸化水平。最后应用ERK1/2抑制剂(PD98059)与TAM共同作用,观察其对胶质瘤细胞内ERK1/2磷酸化水平和细胞凋亡的影响。实验结果显示:TAM可呈浓度和时间依赖性地抑制胶质瘤细胞生长; TAM处理组的细胞凋亡明显增加且呈浓度依赖性;TAM能增加细胞内ERK1/2磷酸化水平;以PD98059阻断ERK1/2的激活,能增强TAM诱导细胞凋亡的作用。实验结果表明TAM能够抑制胶质瘤细胞生长和促进其细胞凋亡, ERK1/2信号通路的激活参与调控TAM所致胶质瘤细胞凋亡。     

沙眼衣原体通过激活MAPK/ERK信号通路抑制宿主细胞凋亡

目的:研究沙眼衣原体抑制宿主细胞凋亡活性与MAPK/ERK信号通路的关系。方法:利用化学抑制剂U0126阻断MAPK/ERK信号通路,然后分别采用流式细胞术、Caspase-3活性检测试剂盒和Western Blot实验检测沙眼衣原体感染细胞在凋亡诱导剂Etoposide作用下细胞凋亡率和Caspase-3活性变化,以及PARP是否发生裂解。结果:当MAPK/ERK信号通路被阻断时,在Etoposide的作用下,沙眼衣原体感染细胞凋亡率明显上升,同时Caspase-3被活化和PARP发生裂解。结论:沙眼衣原体抑制宿主细胞凋亡活性与MAPK/ERK信号通路激活有关。     

Oleanolic Acid Suppresses Migration and Invasion of Malignant Glioma Cells by Inactivating MAPK/ERK Signaling Pathway

Mitogen-activated protein kinases/Extracellular signal-regulated kinase (MAPK/ERK) pathway is essential for migration and invasion of malignant glioma. It is efficient to inhibit migration and invasion of glioma cells by targeting this pathway. Oleanolic acid (OA) has been well demonstrated to suppress survival, growth and angiogenesis of glioma cells. However, it is still unknown if OA affects the migration and invasion of glioma cells. We utilized U-87 MG glioma cell lines and primary glioma cells from patients to study the effect of OA on migration and invasion of glioma cells with multidisciplinary approaches. In this study, we found that OA significantly decreased the ability of glioma cells to migrate and invade. Epithelial-mesenchymal transition (EMT) of glioma cells was also suppressed by OA treatment. Furthermore, MAPK/ERK pathway was greatly inhibited in glioma cells under OA treatment. MAPK/ERK reactivation induced by a recombinant lentiviral vector, Lv-MEK, was able to rescue the inhibitory effect of OA on migration and invasion of glioma cells. Taken together, we provided evidences that OA was a MAPK/ERK pathway-targeting anti-tumor agent. Although the concentrations we used exceeded its physiological level, OA may be used to prevent migration and invasion of glioma cells by developing its derivatives with enhanced bioactivity.     

Lithium Inhibits Tumorigenic Potential of PDA Cells through Targeting Hedgehog-GLI Signaling Pathway

Hedgehog signaling pathway plays a critical role in the initiation and development of pancreatic ductal adenocarcinoma (PDA) and represents an attractive target for PDA treatment. Lithium, a clinical mood stabilizer for mental disorders, potently inhibits the activity of glycogen synthase kinase 3β (GSK3β) that promotes the ubiquitin-dependent proteasome degradation of GLI1, an important downstream component of hedgehog signaling. Herein, we report that lithium inhibits cell proliferation, blocks G1/S cell-cycle progression, induces cell apoptosis and suppresses tumorigenic potential of PDA cells through down-regulation of the expression and activity of GLI1. Moreover, lithium synergistically enhances the anti-cancer effect of gemcitabine. These findings further our knowledge of mechanisms of action for lithium and provide a potentially new therapeutic strategy for PDA through targeting GLI1.     

靶向Wnt信号的溶瘤腺病毒抑制肝癌干细胞研究

肝细胞癌(hepatocellular carcinoma,HCC)是全球第五大癌症并成为癌症死亡的主要原因,传统治疗早期肝癌取得了一定的进展,但是癌症的复发、转移和耐药仍未得到根本解决,这些现象可通过癌症干细胞理论(cancer stem cell,CSC)进行解释.本研究通过悬浮富集培养的方法,获得了MHCC-97H细胞的三维立体球细胞(sphere cell),并检测其干细胞特性,通过删除5型腺病毒的E1A CR2区域24 bp碱基,并用Wnt活性转录元件TCF/TEF调控E1A基因表达,同时插入抗癌基因TSLC1,得到了双靶向溶瘤腺病毒Ad.wnt-E1A(△24 bp)-TSLC1,通过MTT、结晶紫染色实验、Hoechst、细胞划痕、蛋白质印迹技术、Transwell及免疫荧光技术检测重组病毒对肝癌类干细胞的EMT(epithelial-mesenchymal transition)转化、杀伤、凋亡以及迁移的作用.结果表明,悬浮富集培养的MHCC-97H sphere细胞具有自我更新、分化能力、静息性以及耐药性,高表达肝癌干细胞表面标志物(如CD133等),重组病毒处理后表现出明显的杀伤效果及抑制细胞迁移与侵袭的特性,靶向抑制MHCC-97H sphere细胞能力更强(P0.001),且重组病毒能有效诱导肝癌类干细胞通过caspase途径发生凋亡.因此,重组病毒Ad.wnt-E1A(△24 bp)-TSLC1有可能成为靶向肝癌干细胞的治疗药物,具有一定的临床应用前景.     

微波辐射对PC12细胞Raf/MEK/ERK信号通路相关分子表达的影响

体外培养PC12细胞,将其诱导分化为神经元后,建立微波辐射细胞模型,采用免疫印迹技术和图像分析技术研究微波辐射后Raf/MEK/ERK信号通路相关分子的动态表达变化规律,进一步探讨微波辐射损伤的分子机制。结果发现,微波辐射后6h～3d,假辐射组和辐射组PC12细胞中Raf-1、ERK表达均呈先增加后减少趋势,两组差别不显著,但辐射组Raf-1、ERK和CREB的磷酸化水平均较假辐射组明显升高,表明Raf/MEK/ERK信号通路活化增强可能是微波辐射致神经细胞损伤的重要机制。     

VEGFR,RET, and RAF/MEK/ERK Pathway Take Part in the Inhibition of Osteosarcoma MG63 Cells with Sorafenib Treatment

Osteosarcoma (OS) is the leading primary malignant bone tumor in children and young adults. It is response for a high mortality rate. Nowadays, few researches have been performed on sorafenib against OS and no tools are available to guide the use of sorafenib in the OS treatment. In this study, we aim to investigate the effect of sorafenib on OS cell MG63 and figure the potential effective molecular pathway of its function. In the present study, we performed assays of cell proliferation, RT-PCR, and western blot to investigate the effect of sorafenib on OS MG63 cells and to elucidate the molecular actions of sorafenib against RTKs VEGFR2 and RET, as well as MEK/ERK signaling pathway. The present study confirmed that sorafenib could inhibit the proliferation of OS MG63 cells and caused a series of biomolecule effects, including the change of VEGFR2 and ERK gene expression, and the phosphorylation alteration of VEGFR2, RET, and MEK1 proteins. VEGFR2, RET, and MEK/ERK signaling pathway are involved in the pharmacological mechanism of sorafenib. They are potential candidate targets for OS treatment.     

Chlamydial Antiapoptotic Activity Involves Activation of the Raf/MEK/ERK Survival Pathway

Chlamydiae are obligate intracellular bacteria that cause variety of human diseases. Chlamydia-infected host cells are profoundly resistant to apoptosis induced by many different apoptotic stimuli. The inhibition of apoptosis is thought to be an important immune escape mechanism allowing chlamydiae to productively complete their obligate intracellular growth cycle. Infection with chlamydiae can activate the Raf/MEK/ERK pathway. Because the survival pathway can modulate apoptosis, we used MEK-specific inhibitor U0126 and Raf-specific inhibitor GW5074 to examine the role of Raf/MEK/ERK pathway in chlamydial antiapoptotic activity. Apoptosis was induced by staurosporine (STS) and detected by morphology, DNA fragmentation, caspase-3 activation, and poly (ADP-ribose) polymerase cleavage. Inhibition of the pathway sensitized Chlamydia-infected cells to STS-mediated cell apoptosis. The data indicate that chlamydial antiapoptotic activity involves activation of the Raf/MEK/ERK survival pathway.