RAP1-mediated MEK/ERK pathway defects in Kabuki syndrome

The genetic disorder Kabuki syndrome (KS) is characterized by developmental delay and congenital anomalies. Dominant mutations in the chromatin regulators lysine (K)–specific methyltransferase 2D (KMT2D) (also known as MLL2) and lysine (K)–specific demethylase 6A (KDM6A) underlie the majority of cases. Although the functions of these chromatin-modifying proteins have been studied extensively, the physiological systems regulated by them are largely unknown. Using whole-exome sequencing, we identified a mutation in RAP1A that was converted to homozygosity as the result of uniparental isodisomy (UPD) in a patient with KS and a de novo, dominant mutation in RAP1B in a second individual with a KS-like phenotype. We elucidated a genetic and functional interaction between the respective KS-associated genes and their products in zebrafish models and patient cell lines. Specifically, we determined that dysfunction of known KS genes and the genes identified in this study results in aberrant MEK/ERK signaling as well as disruption of F-actin polymerization and cell intercalation. Moreover, these phenotypes could be rescued in zebrafish models by rebalancing MEK/ERK signaling via administration of small molecule inhibitors of MEK. Taken together, our studies suggest that the KS pathophysiology overlaps with the RASopathies and provide a potential direction for treatment design.     

Targeting the ERK signaling pathway in cancer therapy

The extracellular signal‐regulated kinase (ERK) signaling pathway is a major determinant in the control of diverse cellular processes such as proliferation, survival, differentiation and motility. This pathway is often up‐regulated in human tumors and as such represents an attractive target for the development of anticancer drugs. Because of its multiple roles in the acquisition of a complex malignant phenotype, specific blockade of the ERK pathway is expected to result in not only an anti‐proliferative effect but also in anti‐metastatic and anti‐angiogenic effects in tumor cells. Recently potent small‐molecule inhibitors targeting the components of the ERK pathway have been developed. Among them, BAY 43‐9006 (Raf inhibitor), and PD184352, PD0325901 and ARRY‐142886 (MEK1/2 inhibitors) have reached the clinical trial stage. We briefly discuss the possibility that combination of ERK pathway inhibitors (cytostatic agents) and conventional anticancer drugs (cytotoxic agents) provides an excellent basis for the development of new chemotherapeutic strategies against cancer.     

Targeting the ERK signaling pathway in cancer therapy

The extracellular signal-regulated kinase (ERK) signaling pathway is a major determinant in the control of diverse cellular processes such as proliferation, survival, differentiation and motility. This pathway is often up-regulated in human tumors and as such represents an attractive target for the development of anticancer drugs. Because of its multiple roles in the acquisition of a complex malignant phenotype, specific blockade of the ERK pathway is expected to result in not only an anti-proliferative effect but also in anti-metastatic and anti-angiogenic effects in tumor cells. Recently potent small-molecule inhibitors targeting the components of the ERK pathway have been developed. Among them, BAY 43-9006 (Raf inhibitor), and PD184352, PD0325901 and ARRY-142886 (MEK1/2 inhibitors) have reached the clinical trial stage. We briefly discuss the possibility that combination of ERK pathway inhibitors (cytostatic agents) and conventional anticancer drugs (cytotoxic agents) provides an excellent basis for the development of new chemotherapeutic strategies against cancer.     

FOXA1 overexpression suppresses interferon signaling and immune response in cancer

Androgen receptor–positive prostate cancer (PCa) and estrogen receptor–positive luminal breast cancer (BCa) are generally less responsive to immunotherapy compared with certain tumor types such as melanoma. However, the underlying mechanisms are not fully elucidated. In this study, we found that FOXA1 overexpression inversely correlated with interferon (IFN) signature and antigen presentation gene expression in PCa and BCa patients. FOXA1 bound the STAT2 DNA-binding domain and suppressed STAT2 DNA-binding activity, IFN signaling gene expression, and cancer immune response independently of the transactivation activity of FOXA1 and its mutations detected in PCa and BCa. Increased FOXA1 expression promoted cancer immuno- and chemotherapy resistance in mice and PCa and BCa patients. These findings were also validated in bladder cancer expressing high levels of FOXA1. FOXA1 overexpression could be a prognostic factor to predict therapy resistance and a viable target to sensitize luminal PCa, BCa, and bladder cancer to immuno- and chemotherapy.     

LncRNA UCA1介导Raf/MEK/ERK信号通路调控乳腺癌细胞生物学作用的机制研究

目的 探讨长链非编码RNA(long non-coding RNA, LncRNA)尿路上皮癌胚抗原1(urothelial carcinoma antigen1,UCA1)在乳腺癌细胞中的表达及其对乳腺癌细胞生物学作用的机制研究。方法 实时定量聚合酶链式反应(quantitative real-time PCR, qRT-PCR)法检测LncRNA UCA1在正常人乳腺上皮细胞MCF-10A与乳腺癌细胞BT-474,MCF-7,SKBR-3和MDA-MB453中的表达;选择BT-474和MCF-7细胞随机分为三组,分别转染UCA1-siR,NC-siR及Control,再通过qRT-PCR法验证转染后BT-474和MCF-7细胞中LncRNA UCA1表达;通过CCK-8法、Transwell实验和流式细胞仪检测BT-474和MCF-7细胞增殖、侵袭、凋亡能力;利用Western blot检测两种细胞中RAF/MEK/ERK通路相关蛋白的表达。结果 与MCF-10A相比,LncRNA UCA1在BT-474,MCF-7, SKBR-3和MDA-MB453细胞中表达水平分别上调了133...     

Rat parathyroid hormone 1-34 signals through the MEK/ERK pathway to induce cardiac hypertrophy

This study aimed to characterize the role of the mitogen-activated protein kinase (MAPK) kinase (MEK)/extracellular signal-regulated kinase (ERK) pathway in cardiac hypertrophy induced by parathyroid hormone (PTH). Various concentrations of rat PTH1-34 were used to induce hypertrophy in neonatal rat ventricular cardiomyocytes, and the effects were compared with control cells and those treated with PD98059, a selective inhibitor of MEK1. Hypertrophy was assessed in terms of cell diameter, atrial natriuretic peptide (ANP) mRNA expression and protein synthesis; the MEK/ERK pathway was assessed by measuring levels of phosphorylated ERK1/2. Treatment with PTH1-34 at 100 nM for 24 h effectively induced cardiac hypertrophy (increased cell diameter, protein synthesis and ANP mRNA expression) and also increased levels of phosphorylated ERK1/2 compared with normal control cells. Treatment with PTH1-34 plus PD98059 significantly attenuated these changes. These results demonstrate that inhibition of the MEK/ERK pathway blocks PTH1-34-induced cardiac hypertrophy, suggesting that PTH1-34 might signal through the MAPK pathway to induce hypertrophy in cardiomyocytes.     

RAS/MAPK通路在食管癌中的活化改变及其意义

目的通过研究细胞外信号调节激酶（ERK）在食管癌组织、癌旁组织、正常组织中的表达和激活情况,探讨RAS／MAPK信号级联在食管癌发生中的意义。方法Western—blot方法检测15例新鲜食管癌组织及相应的13例癌旁组织、14例正常组织中p-ERK、ERK的表达。免疫组化方法检测该批组织石蜡切片中p-ERK的表达及其细胞定位。结果Western—blot显示全部组织均表达ERK和p-ERK,统计分析表明,ERK在3种组织中的表达差异无统计学意义（P〉0．05）;p-ERK在正常组织、癌旁组织、癌组织中的表达有下降趋势,其在癌组织中的表达显著低于正常组织（P〈0．05）。免疫组化则显示全部组织均表达p-ERK,阳性信号主要位于食管黏膜上皮细胞和癌细胞的胞核。结论RAS／MAPK信号转导通路的活化存在于所有食管癌组织、癌旁组织和正常组织,食管癌中该通路的活化水平显著低于正常组织,该通路活化水平的改变可能与食管癌的发生有关。     

Endothelin signaling in osteoblasts: global genome view and implication of the calcineurin/NFAT pathway

Patients with prostate cancer develop osteoblastic metastases when tumor cells arrive in the bone and stimulate osteoblasts by secreting growth-promoting factors. Endothelin 1 (ET-1) is believed to be a key factor in promoting osteoblastic metastasis. Selective blockade of the ET(A) receptor is an established strategy in the development of cancer therapeutics. However, the molecular mechanisms whereby prostate cancer promotes abnormal bone growth are not fully understood. In this study, we have applied genomic approaches to elucidate the molecular mechanism of stimulation of osteoblasts by ET-1. To examine the ET-1 axis, we generated genomic signatures for osteoblasts treated with ET-1, in the presence and absence of a selective ET(A) antagonist (ABT-627). The ET-1 signature was comprised of several motifs, such as osteoblastic differentiation, invasion, and suppression of apoptosis. The signature also pointed at possible activation of the calcineurin/NFAT pathway. We showed that ET-1 activates calcineurin and causes nuclear translocation of NFATc1, implicating the pathway in the ET-1-mediated stimulation of osteoblasts. We also showed that ET-1 inhibits apoptosis in osteoblasts, implying that the suppression of apoptosis may be an important factor in the promotion of osteoblastic growth by ET-1.     

HMGA1 facilitates tumor progression through regulating Wnt/β-catenin pathway in endometrial cancer

Recent studies have identified a unique role for high mobility group protein A1 (HMGA1) as a major regulator of tumor progression and in diverse tumor models. Emerging evidences indicate that overexpressed HMGA1 facilitates multiple malignant phenotypes of cancer cells, however, the oncogenic activities of HMGA1 in endometrial cancer (EC) remains elusive. Here we showed that HMGA1 was more frequently expressed in human EC tissues compared to non-tumor tissues. Elevated HMGA1 was significantly associated with advanced clinical stage. Wound-healing assay and transwell assay showed that HMGA1 can positively regulate cell migration and invasion. Mechanistically, luciferase reporter assay and Western blotting assay demonstrated that activation of Wnt/β-catenin pathway contributed to the oncogenic activity of HMGA1. Taken together, our data reveal that HMGA1 may function as an oncogene and modulate EC cell migration and invasion by activating Wnt/β-catenin pathway, implying that suppression of HMGA1 might be a potential therapeutic strategy for EC.     

ERKl／2信号通路在高糖诱导的HK-2上皮间质转分化中的作用

目的：观察高糖诱导的细胞外信号调节激酶（ERK1/2）信号通路活化在上皮间质转分化中的作用,从而探讨延缓糖尿病肾病肾间质纤维化的发生机制。方法体外培养HK-2细胞,随机分为正常对照组、高糖组、ERK1/2通路抑制剂PD98059＋高糖组和高渗组,处理72 h后收集细胞,应用免疫细胞化学方法检测α-SMA、CK18的表达;应用Western blot法检测磷酸化ERK1/2、总ERK1/2表达水平的变化。结果（1）正常对照组胞质有大量CK18蛋白阳性表达,而α-SMA蛋白表达呈阴性;高糖组胞质中可见大量α-SMA蛋白强阳性染色,而CK18蛋白呈阴性表达;经PD98059处理后,CK18表达较高糖组染色深,而α-SMA表达较高糖组染色浅;高渗组胞质中CK18呈阳性表达,α-SMA表达呈阴性。（2）正常对照组可见少量总ERK1/2表达,p-ERK1/2蛋白仅有微量表达;经高糖刺激48 h后,总ERK1/2表达较正常对照组无明显差异,p-ERK1/2蛋白表达明显增加（P＜0.05）;而使用PD98059处理后,总ERK1/2的变化不大,但p-ERK1/2蛋白的表达却显著降低（P＜0.05）;高渗组与正常对照组无显著差异。结论 ERK1/2信号通路可能参与了高糖诱导的人近端肾小管上皮细胞（HK-2）转分化过程,阻断 ERKl/2可部分抑制人近端肾小管上皮细胞的转分化,进而延缓肾小管间质纤维化的发生和发展。     

碱性成纤维细胞生长因子对人涎腺腺样囊性癌ACC-2细胞株增殖及MEK/ERK、MKP-1通路的影响

目的:观察碱性成纤维细胞生长因子(basic fibroblast growth factor,bFGF)对人涎腺腺样囊性癌ACC-2细胞株增殖及MEK1/2、ERK1/2及MKP-1表达的影响。方法:培养人涎腺腺样囊性癌细胞株(ACC-2),MTT比色法测定不同浓度bFGF对细胞增殖的影响;免疫沉淀法纯化蛋白并ERK试剂盒测定ERK活性;免疫印迹法测定p-MEK1/2、p-ERK1/2及MKP-1表达。结果:MTT实验显示bFGF明显增强ACC-2细胞增殖,免疫沉淀法显示bFGF上调ERK活性,免疫印迹法显示bFGF明显增强p-MEK1/2、p-ERK1/2表达及抑制MKP-1表达。结论:bFGF可促进人涎腺腺样囊性癌ACC-2细胞株增殖,其途径与上调ERK活性、激活MEK/ERK通路、抑制MKP-1表达有关。     

肠道病毒71型激活人横纹肌肉瘤细胞中JNK1/2信号转导通路

目的探讨c-Jun N端激酶(JNK)信号转导通路在肠道病毒71型(EV71)感染中的作用。方法台盼蓝染色法检测不同浓度抑制剂SP600125对人横纹肌肉瘤细胞(RD)活性的影响;实时荧光定量PCR及western blot检测EV71感染RD细胞后VP1 mRNA及蛋白质的表达水平;western blot检测JNK1/2、c-Fos、c-Jun蛋白及其磷酸化水平,并分析JNK1/2抑制剂SP600125对EV71复制及JNK1/2信号通路的影响。结果台盼蓝染色结果表明,与空白对照组比较,5和10μmol/L实验组存活率差异无统计学意义(P均0.05),而20μmol/L抑制剂组细胞存活率明显降低(P0.05);实时荧光定量PCR及western blot结果表明,与对照组相比,实验组在EV71感染RD细胞8 h后,其VP1 mRNA及蛋白质的表达水平均明显下降(P均0.01)。此外,western blot检测结果证实EV71感染RD细胞后,其JNK1/2、c-Fos和c-Jun的蛋白质磷酸化水平均明显升高(P均0.05)。而经抑制剂SP600125处理可明显下调其JNK1/2、c-Fos、c-Jun磷酸化水平(P均0.05)。结论 JNK1/2信号通路可被EV71感染有效激活,并与EV71复制有关。     

Th1 cytokine interferon gamma improves response in HER2 breast cancer by modulating the ubiquitin proteasomal pathway

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Quercetin nanoparticles induced autophagy and apoptosis through AKT/ERK/Caspase-3 signaling pathway in human neuroglioma cells: In vitro and in vivo

Neuroglioma is a complex neuroglial tumor involving dysregulation of many biological pathways at multiple levels. Quercetin is a potent cancer therapeutic agent presented in fruit and vegetables, preventing tumor proliferation, and is a well known cancer therapeutic agent and autophagy mediator. Recent studies showed that drug delivery by nanoparticles have enhanced efficacy with reduced side effects. In this regard, gold-quercetin into poly (dl-lactide-co-glycolide) nanoparticles was examined. In the present study, quercetin nanoparticle induced cell autophagy and apoptosis in human neuroglioma cell was investigated. Quercetin nanoparticle administrated to animals displayed suppressed role in tumor growth. The cell viability was deterined through CCK8 assay. Transmission electron microscopy was utilized to observe the formation of autophagosome. The cell apoptosis was assessed by annexin V-PI staining. The protein expression of cell autophagy regulators and tumor suppressors were analyzed via western blot and RT-PCR. Treatment of human neuroglioma cell with quercetin nanoparticle induced cell death in a dose-and time-dependent manner. The flow cytometry results showed that the proportion of the apoptosis cells had gained after quercetin nanoparticle treatment compared to untreatment group. Moreover, the expression of activated PI3K/AKT and Bcl-2 were down-regulated upon quercetin nanoparticle treatment in human neuroglioma cells. The expression level of LC3 and ERK as well as cytoplasm p53, cleaved Caspase-3 and PARP was positively correlated with the concentration of quercetin nanoparticle. In addition, p-mTOR and GAIP were obviously down-regulated by quercetin nanoparticle treatment in a dose-dependent manner. These results indicated that quercetin nanoparticle could induce autophagy and apoptosis in human neuroglioma cells, the underlying molecular mechanisms, at least partly, through activation LC3/ERK/Caspase-3 and suppression AKT/mTOR signaling.     

Evidence for defective transmembrane signaling in B cells from patients with Wiskott-Aldrich syndrome.

B lymphocytes from patients expressing the X chromosome-linked immune deficiency disorder, Wiskott-Aldrich syndrome (WAS), fail to produce antibodies in response to stimulation with polysaccharides and other type-2 T cell-independent antigens. To investigate whether this abnormality reflects a defect in the signal transduction cascade normally triggered by ligation of surface immunoglobulin (sIg) on B cells, we have examined early signaling events induced by anti-Ig antibody stimulation of EBV B lymphoblastoid cell lines from WAS patients and healthy controls. Despite the expression of comparable levels of sIg and sIgM on WAS and control EBV B cells, WAS cells failed to manifest the increased proliferation in response to anti-Ig treatment observed in the control cell lines. WAS and control EBV B cells also differed in the magnitude of the change in cytosolic free calcium ([Ca2+]i) induced by sIg ligation; WAS cells showed either markedly diminished or no changes in [Ca2+]i levels whereas control EBV B cells consistently showed increases in [Ca2+]i. Anti-Ig-induced changes in inositol phosphate release were also markedly reduced in WAS compared with control cells. As protein tyrosine phosphorylation is thought to represent a proximal event in the activation of B cells, inducing increases in [Ca2+]i by virtue of tyrosine phosphorylation of phospholipase C (PLC)-gamma, profiles of protein tyrosine phosphorylation and expression of tyrosine-phosphorylated PLC-gamma 1 were compared between WAS and normal EBV B cells before and after sIg cross-linking. These studies revealed that in addition to defective mobilization of Ca2+, the WAS cells manifested little or no increase in tyrosine phosphorylation of PLC-gamma 1 or other intracellular proteins after sIg ligation. Together these results indicate the association of WAS with a defect in the coupling of sIg to signal transduction pathways considered prerequisite for B cell activation, likely at the level of tyrosine phosphorylation. The abnormalities observed in these early transmembrane signaling events in WAS EBV B cells may play a role not only in the nonresponsiveness of WAS patient B cells to certain T independent antigens, but also in the genesis of some of the other cellular deficits exhibited by these patients.     

Notch1信号通路在乳腺癌中的研究进展

乳腺癌是女性最常见、死亡率最高的恶性肿瘤之一,近年来其发病率不断升高。研究发现Notch1信号通路在乳腺癌中起着重要的作用。研究发现Notch1在正常乳腺组织和乳腺良性病变以及乳腺癌中均有表达,在乳腺浸润性导管癌中的表达率明显高于导管原位癌,其表达与乳腺癌HER-2亚型有关,在HER-2阳性型中的Notch1的阳性表达率明显高于其他亚型。研究认为Notch1阳性表达率高与其乳腺癌的分化程度低、分期晚、淋巴结转移阳性率高以及脑转移有关,预示着乳腺癌的预后差,并被认为可作为判断预后的临床指标。此外研究发现Notch1的高表达可导致乳腺癌对阿霉素与紫杉醇的耐药。研究发现Notch1高表达与乳腺癌侵袭强、凋亡抑制有关。研究还发现Notch1的表达影响乳腺癌干细胞的数量,是维持乳腺癌干细胞恶性表型的重要因子。且Notch1与VEGF、c-myc、CCL2、TRB3、MMP2等相关基因表达及肿瘤微环境相关。针对Notch1的靶向治疗的研究有Notch1单克隆抗体、三氧化二砷以及金雀异黄素等药物,以及Notch1相关通路的靶向治疗药物,均可抑制乳腺癌细胞的生长,对乳腺癌有抗肿瘤作用。     

Regulation of GST-MDA-7 toxicity in human glioblastoma cells by ERBB1, ERK1/2, PI3K, and JNK1-3 pathway signaling

The present studies defined the biological effects of a GST fusion protein of melanoma differentiation-associated gene-7 (mda-7), GST-MDA-7 (1 and 30 nmol/L), on cell survival and cell signaling in primary human glioma cells in vitro. GST-MDA-7, in a dose- and time-dependent fashion killed glioma cells with diverse genetic characteristics; 1 nmol/L caused arrest without death, whereas 30 nmol/L caused arrest and killing after exposure. Combined inhibition of extracellular signal-regulated kinase 1/2 (ERK1/2) and AKT function was required to enhance 1 nmol/L GST-MDA-7 lethality in all cell types, whereas combined activation of MEK1 and AKT was required to suppress 30 nmol/L GST-MDA-7 lethality; both effects are mediated in part by modulating c-Jun NH(2)-terminal kinase (JNK) 1-3 activity. The geldanamycin 17AAG inhibited AKT and ERK1/2 in GBM cells and enhanced GST-MDA-7 lethality. JNK1-3 signaling promoted BAX activation and mitochondrial dysfunction. In GBM6 cells, GST-MDA-7 (30 nmol/L) transiently activated p38 mitogen-activated protein kinase, which was modestly protective against JNK1-3-induced toxicity, whereas GST-MDA-7 (300 nmol/L) caused prolonged intense p38 mitogen-activated protein kinase activation, which promoted cell death. In GBM12 cells that express full-length mutant activated ERBB1, inhibition of ERBB1 did not modify GST-MDA-7 lethality; however, in U118 established glioma cells, stable overexpression of wild-type ERBB1 and/or truncated active ERBB1vIII suppressed GST-MDA-7 lethality. Our data argue that combined inhibition of ERK1/2 and AKT function, regardless of genetic background, promotes MDA-7 lethality in human primary human glioma cells via JNK1-3 signaling and is likely to represent a more ubiquitous approach to enhancing MDA-7 toxicity in this cell type than inhibition of ERBB1 function.