Trop2 expression contributes to tumor pathogenesis by activating the ERK MAPK pathway

Background  

Trop2 is a cell-surface glycoprotein overexpressed by a variety of epithelial carcinomas with reported low to restricted expression in normal tissues. Expression of Trop2 has been associated with increased tumor aggressiveness, metastasis and decreased patient survival, but the signaling mechanisms mediated by Trop2 are still unknown. Here, we studied the effects murine Trop2 (mTrop2) exerted on tumor cellular functions and some of the signaling mechanisms activated by this oncogene.     

Integrating signals from RTKs to ERK/MAPK

Signals received at the cell surface must be properly transmitted to critical targets within the cell to achieve the appropriate biological response. This process of signal transduction is often initiated by receptor tyrosine kinases (RTKs), which function as entry points for many extracellular cues and play a critical role in recruiting the intracellular signaling cascades that orchestrate a particular response. Essential for most RTK-mediated signaling is the engagement and activation of the mitogen-activated protein kinase (MAPK) cascade comprised of the Raf, MEK and extracellular signal-regulated kinase (ERK) kinases. For many years, it was thought that signaling from RTKs to ERK occurred only at the plasma membrane and was mediated by a simple, linear Ras-dependent pathway. However, the limitation of this model became apparent with the discovery that Ras and ERK can be activated at various intracellular compartments, and that RTKs can modulate Ras/ERK signaling from these sites. Moreover, ERK scaffolding proteins and signaling modulators have been identified that play critical roles in determining the strength, duration and location of RTK-mediated ERK signaling. Together, these factors contribute to the diversity of biological responses generated by RTK signaling.     

Galectin-3 induces cell migration via a calcium-sensitive MAPK/ERK1/2 pathway

The presence and level of circulating galectin-3 (Gal-3), a member of the galectin family, is associated with diverse diseases ranging from heart failure, immune disorders to cancer metastasis and serves as a biomarker of diagnosis and treatment response. However, the mechanisms by which exogenous Gal-3 affects pathobiology events remain elusive. In the current study, we found that exogenous Gal-3 slightly delays, while prolonging tyrosine phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) in HeLa cells through a calcium-sensitive and PKC-dependent signaling pathway. The activation was dependent on the sugar-binding properties of Gal-3, since the antagonist lactose could inhibit it. The sugar-binding motif of Gal-3 was required for the activation of ERK1/2. The activation of ERK1/2 was necessary for the initiation and induction of cell migration associated with the phosphorylation of paxillin. All the results presented in this study suggest a novel calcium-sensitive and PKC-dependent pathway through which circulating Gal-3 promotes cell migration and activating the ERK1/2. Taken together, the data depicted here propose a biological function and a target for the diseases'' associated circulating Gal-3.     

Epiregulin enhances tumorigenicity by activating the ERK/MAPK pathway in glioblastoma

Background

Glioblastoma multiforme (GBM) is one of the most aggressive human tumors, and the establishment of an effective therapeutic reagent is a pressing priority. Recently, it has been shown that the tumor tissue consists of heterogeneous components and that a highly aggressive population should be the therapeutic target.

Methods

Through a single subcutaneous passage of GBM cell lines LN443 and U373 in mice, we have developed highly aggressive variants of these cells named LN443X, U373X1, and U373X2, which showed increased tumor growth, colony-forming potential, sphere-forming potential, and invasion ability. We further investigated using microarray analysis comparing malignant cells with their parental cells and mRNA expression analysis in grades II to IV glioma samples.

Results

Adipocyte enhancer binding protein 1, epiregulin (EREG), and microfibrillar associated protein 5 were identified as candidate genes associated with higher tumor grade and poor prognosis. Immunohistochemical analysis also indicated a correlation of a strong expression of EREG with short overall survival. Furthermore, both EREG stimulation and EREG introduction of GBM cell lines were found to increase phosphorylation of epidermal growth factor receptor (EGFR) and extracellular signal-regulated kinase and resulted in the promotion of colony formation, sphere formation, and in vivo tumor formation. Gefitinib treatment inhibited phosphorylation of EGFR and extracellular signal-regulated kinase and led to tumor regression in U373-overexpressed EREG.

Conclusion

These results suggested that EREG is one of the molecules involved in glioma malignancy, and EGFR inhibitors may be a candidate therapeutic agent for EREG-overexpressing GBM patients.     

Procaine Inhibits the Proliferation and Migration of Colon Cancer Cells Through Inactivation of the ERK/MAPK/FAK Pathways by Regulation of RhoA

Colon cancer is one of the most lethal varieties of cancer. Chemotherapy remains as one of the principal treatment approaches for colon cancer. The anticancer activity of procaine (PCA), which is a local anesthetic drug, has been explored in different studies. In our study, we aimed to explore the anticancer effect of PCA on colon cancer and its underlying mechanism. The results showed that PCA significantly inhibited cell viability, increased the percentage of apoptotic cells, and decreased the expression level of RhoA in HCT116 cells in a dose-dependent manner (p < 0.05 or p < 0.01). Moreover, PCA increased the proportion of HCT116 cells in the G₁ phase as well as downregulated cyclin D1 and cyclin E expressions (p < 0.05). In addition, we found that PCA remarkably inhibited cell migration in HCT116 cells (p < 0.01). However, all these effects of PCA on cell proliferation, apoptosis, and migration were significantly reversed by PCA + pc-RhoA (p < 0.05 or p < 0.01). PCA also significantly decreased the levels of p-ERK, p-p38MAPK, and p-FAK, but PCA + pc-RhoA rescued these effects. Furthermore, the ERK inhibitor (PD098059), p38MAPK inhibitor (SB203580), and FAK inhibitor (Y15) reversed these results. These data indicate that PCA inhibited cell proliferation and migration but promoted apoptosis as well as inactivated the ERK/MAPK/FAK pathways by regulation of RhoA in HCT116 cells.     

Phosphatidylinositol 3'-kinase and MAPK/ERK kinase 1/2 differentially regulate expression of vascular endothelial growth factor in human malignant astrocytoma cells

Malignant astrocytomas are characterized by extensive vascularization attributed to increased expression of the angiogenic cytokine vascular endothelial growth factor (VEGF). VEGF is elevated in astrocytomas under normal oxygen conditions and undergoes induction in hypoxic stress. Prior studies have shown that both the phosphatidylinositol 3'-kinase (PI3-kinase) and MEK1/2 (MAPK/ERK kinase 1/2) pathways promote proliferation of astrocytoma cells and growth of astrocytic tumors. Whether these pathways regulate growth by modulating angiogenesis as well as proliferation is not clear. In this study, pharmacologic inhibitors were used to specifically inhibit PI3-kinase and MEK1/2 activity in human malignant astrocytoma cell lines, and their effects on VEGF expression were determined. Northern blot analysis of VEGF messenger RNA (mRNA) from cells treated with inhibitors demonstrated cell line-specific responses. The PI3-kinase pathway regulated both the normoxic expression and hypoxic induction of VEGF in 2 cell lines, whereas MEK1/2 regulated only the normoxic expression in the same 2 lines. The third cell line showed no change in VEGF mRNA with inhibition of either of these 2 pathways. This study suggests that modulation of signaling pathways implicated in proliferation of astrocytoma cell lines may have varying effects in vivo depending on the role these pathways play in regulating tumor angiogenesis.     

MAPK/ERK 信号通路在肺癌耐药中的研究进展

耐药性的出现是肺癌临床药物治疗失败的主要原因,其机制与相关信号通路的异常活化密切相关.研究发现,MAPK/ERK信号途径作为细胞增殖、分化、凋亡、黏附和迁移等胞内信号传导的重要通路之一,其异常激活也是引发肺癌化疗、靶向、免疫等治疗药物产生耐药的关键所在,并有望成为逆转肺癌耐药新的干预靶点.全文主要就MAPK/ERK信号...     

EGF调节MT1-MMP和MMP-2的表达及其信号传导机制的研究

目的:探讨表皮生长因子（EGF）在宫颈癌SiHa细胞中对膜型基质金属蛋白酶（MT1-MMP）和基质金属蛋白酶-2（MMP-2）表达的调节作用,明确其相关的信号传导机制。方法:利用EGF作为干预因素,信号通路阻断剂分别阻断表皮生长因子受体（EGFR）、AKT、ERK、p38和JNK的磷酸化,来观察EGF对MT1-MMP和MMP-2表达的影响及相关信号通路。结果:EGF在mRNA和蛋白水平上诱导MT1-MMP的表达增加和抑制MMP-2的表达;MAPK和ERK激酶抑制剂在mRNA和蛋白水平上可阻断这种诱导;PI3-K抑制剂不影响EGF对MT1-MMP的诱导,但可进一步抑制MMP-2的表达。结论:EGFR通过MAPK/ERK信号通路上调MP1-MMP的表达和下调MMP-2的表达,同时还通过PI3-K/AKT的信号通路轻度上调MMP-2的表达。此外,EGF可以提高细胞培养基中MMP-2的活性。     

ERK/MAPK信号传导途径在乳腺肿瘤治疗中的意义

ERK／MAPK途径在乳腺癌的发生和发展中起着重要的作用，该通路有3个关键靶分子：小G蛋白Ras及其下游的Raf激酶、MEK1／2和ERK1／2。ERK／MAPK信号转导途径的激活将促进乳腺肿瘤细胞的增殖，因此阻断该信号转导途径就可以干预肿瘤的进程，这为乳腺癌的治疗提供了一种新的策略。目前主要有3种抑制ERK／MAPK信号转导途径的方法：（1）利用抑制剂破坏主要靶蛋白的结构和功能；（2）抑制靶蛋白之间的相互作用，阻止信号转导；（3）利用反义核苷酸技术使靶分子功能缺失从而阻断级联反应。这些方法为乳腺肿瘤的治疗提供了新的思路，相信它们将对乳腺肿瘤的治疗做出重要的贡献。     

Post-translational regulation of the ERK phosphatase DUSP6/MKP3 by the mTOR pathway

MAP kinases phosphatases (MKPs) belong to the dual-specificity phosphatase family (DUSP) and dephosphorylate phosphothreonine and phosphotyrosine within MAP kinases. We had previously shown that DUSP6/MKP-3 was phosphorylated and degraded upon growth factor stimulation, in a MEK-dependent manner. Here we show that another pathway involved in growth factor signaling, the PI3K/mTOR signaling pathway, accounts for a part of the phosphorylation and degradation of DUSP6 induced by serum growth factors, as evidenced by experiments using pharmacological inhibitors of PI3 kinase and mammalian target of rapamycin (mTOR). Moreover, specific agonists of the mTOR pathway, such as amino acids or insulin/IGF-1, which do not activate extracellular signal regulated kinases (ERKs) in our cellular model, were also able to induce the phosphorylation and degradation of DUSP6. However, a basal activity of MEK was required for the mTOR pathway-mediated phosphorylation to occur. Mutagenesis studies identified serine 159 within DUSP6 as the target of the mTOR pathway. The ERK phosphatase DUSP6 may thus constitute a novel branch-point of the crosstalk between two major signaling pathways induced by growth factors, the MEK/ERK pathway and the PI3K/mTOR pathway.     

SIGNIFICANCE AND CORRELATION OF MAPK/ERK2 AND PI3-K IN HUMAN BREAST TUMORIGENESIS

Objective： MAPK （（Mitogen-actived Protein Kinase） and PI3-K （Phosphatidylinositol 3-kinase） pathways have been implicated in the mitogenic pathways regulating cell growth, proliferation, differentiation and transformation and thus involved in tumorigenesis. This study was designed to examined the protein expression, activity and mRNA levels of both ERK and PI3-K in a series of breast tumors and adjacent mammary glands, and to figure out the changes of ERK2 and PI3-K during the dynamic process of breast tumorigenesis. Methods： A series of breast tumors and adjacent mammary glands were collected at surgery, including 37 cases of breast cancer, 6 cases of atypical hyperplasia-breast carcinoma in situ and 15 cases of benign conditions. Western blot, kinase activity assay and RT-PCR were used to detect the protein expression, kinase activity and mRNA level, respectively. Results： The revels of protein, activity and mRNA of ERK2 were elevated during the stages of both initiation and progression. The increasing tendency in breast cancer was equal to atypical hyperplasia -in situ carcinoma, but higher than in benign lesion and adjacent normal mammary gland. PI3-K was activated during the stage of progression of breast cancer. An inverse correlation between the activity of PI3-K and ERK2 in breast cancer was found. Conclusion： Our findings indicate that ERK2 may perform its function during both the stages of breast cancer initiation and breast cancer progression, while PI3-K may exert its effect during the stage of breast cancer progression. Both PI3-k and ERK2 are involved in the tumorigenesis of breast cancer.     

miR-524-5p suppresses the growth of oncogenic BRAF melanoma by targeting BRAF and ERK2

It has been well documented that miRNAs can modulate the effectiveness of cancer-associated signaling pathways. Mitogen-activated protein kinase (MAPK/ERK) signaling plays an essential role in the progression of many cancers, including melanoma and colon cancers. However, no single miRNA is reported to directly target multiple components of the MAPK/ERK pathway. We performed a miRNA PCR array screening with various MAPK/ERK signaling activities. The miRNA array data revealed that the expression of miR-524-5p was decreased in cells with an active MAPK/ERK pathway and confirmed that the expression of miR-524-5p is inversely associated with the activity of the MAPK/ERK pathway. We demonstrated that miR-524-5p directly binds to the 3′-untranslated regions of both BRAFandERK2 and suppresses the expression of these proteins. Because BRAF and ERK2 are the main components of MAPK signaling, the overexpression of miR-524-5p effectively inhibits MAPK/ERK signaling, tumor proliferation, and melanoma cell migration. Moreover, tumors overexpressing miR-524-5p were significantly smaller than those of the negative control mice. Our findings provide new insight into the role of miR-524-5p as an important miRNA that negatively regulates the MAPK/ERK signaling pathway, suggesting that miR-524-5p could be a potent therapeutic candidate for melanoma treatment.     

Interfering with ITGB1-DT expression delays cancer progression and promotes cell sensitivity of NSCLC to cisplatin by inhibiting the MAPK/ERK pathway

Long non-coding RNA ITGB1-DT is involved in the regulation of cancer growth and metastasis. However, the roles of ITGB1-DT in non-small cell lung cancer (NSCLC) progression and sensitivity to cisplatin has not been elucidated. ITGB1-DT expression in NSCLC tissues, and the relationship between ITGB1-DT expression with NSCLC diagnosis, prognosis, clinicopathological features, and immune cell infiltration were investigated in The Cancer Gene Atlas (TCGA) database. The roles and mechanisms of ITGB1-DT in cell growth, migration, and drug sensitivity of NSCLC cells were explored in the cell model. The prognostic nomograms of ITGB1-DT-related genes were evaluated using bioinformatics. ITGB1-DT was overexpressed in NSCLC. Elevated ITGB1-DT expression was related to the late T stage, N stage, M stage, short overall survival (OS), disease-specific survival (DSS), and progression-free interval (PFI) of NSCLC patients. ITGB1-DT was the independent risk factors for poor prognosis, and had diagnostic value for NSCLC patients. Interfering with the ITGB1-DT expression can inhibit the proliferation, migration, and invasion of A549, H1299, and drug-resistant A549/DDP, possibly due to the inhibition of p38 MAPK and ERK phosphorylation levels. ITGB1-DT expression was correlated with the levels of NSCLC immune infiltration cells, such as the TReg, Th, and NK cells. ITGB1-DT-related gene nomograms were associated with the prognosis, and were expected to evaluate the prognosis of NSCLC patients. In conclusion, inhibition of ITGB1-DT expression delayed the growth and metastasis of NSCLC using the MAPK/ERK signaling mechanism and enhanced the sensitivity of NSCLC to cisplatin drugs. These results indicate that ITGB1-DT might be a biomarker for evaluating the diagnosis and prognosis of NSCLC patients.     

Linc-RoR promotes MAPK/ERK signaling and confers estrogen-independent growth of breast cancer

Background

The conversion from estrogen-dependent to estrogen-independent state of ER+ breast cancer cells is the key step to promote resistance to endocrine therapies. Although the crucial role of MAPK/ERK signaling pathway in estrogen-independent breast cancer cell growth is well established, the underlying mechanism is not fully understood.

Methods

In this study, we profiled lncRNA expression against a focused group of lncRNAs selected from lncRNA database. CRISPR/Cas9 was employed to knockout (KO) linc-RoR in MCF-7 cells, while rescue experiments were carried out to re-express linc-RoR in KO cells. Colony formation and MTT assays were used to examine the role of linc-RoR in estrogen-independent growth and tamoxifen resistance. Western blot and qRT-PCR were used to determine the change of protein and lncRNA levels, respectively. The expression of DUSP7 in clinical specimens was downloaded from Oncomine (www.oncomine.org) and the dataset from Kaplan-Meier Plotter (http://kmplot.com) was used to analyze the clinical outcomes in relation to DUSP7.

Results

We identified that linc-RoR functions as an onco-lncRNA to promote estrogen-independent growth of ER+ breast cancer. Under estrogen deprivation, linc-RoR causes the upregulation of phosphorylated MAPK/ERK pathway which in turn activates ER signaling. Knockout of linc-RoR abrogates estrogen deprivation-induced ERK activation as well as ER phosphorylation, whereas re-expression of linc-RoR restores all above phenotypes. Moreover, we show that the ERK-specific phosphatase Dual Specificity Phosphatase 7 (DUSP7), also known as MKP-X, is involved in linc-RoR KO-induced repression of MAPK/ERK signaling. Interestingly, linc-RoR KO increases the protein stability of DUSP7, resulting in repression of ERK phosphorylation. Clinical data analysis reveal that DUSP7 expression is lower in ER+ breast cancer samples than that in ER- breast cancer. Moreover, downregulation of DUSP7 expression is associated with poor patient survival.

Conclusion

Taken together, these results suggest that linc-RoR promotes estrogen-independent growth and activation of MAPK/ERK pathway of breast cancer cells by regulating the ERK-specific phosphatase DUSP7. Thus, this study might help not only in establishing a role for linc-RoR in estrogen-independent and tamoxifen resistance of ER+ breast cancer, but also suggesting a link between linc-RoR and MAPK/ERK pathway.

     

Combination of BMI1 and MAPK/ERK inhibitors is effective in medulloblastoma

BackgroundEpigenetic changes play a key role in the pathogenesis of medulloblastoma (MB), the most common malignant pediatric brain tumor.MethodsWe explore the therapeutic potential of BMI1 and MAPK/ERK inhibition in BMI1^High;CHD7^Low MB cells and in a preclinical xenograft model.ResultsWe identify a synergistic vulnerability of BMI1^High;CHD7^Low MB cells to a combination treatment with BMI1 and MAPK/ERK inhibitors. Mechanistically, CHD7-dependent binding of BMI1 to MAPK-regulated genes underpins the CHD7-BMI1-MAPK regulatory axis responsible of the antitumour effect of the inhibitors in vitro and in a preclinical mouse model. Increased ERK1 and ERK2 phosphorylation activity is found in BMI1^High;CHD7^Low G4 MB patients, raising the possibility that they could be amenable to a similar therapy.ConclusionsThe molecular dissection of the CHD7-BMI1-MAPK regulatory axis in BMI1^High;CHD7^Low MB identifies this signature as a proxy to predict MAPK functional activation, which can be effectively drugged in preclinical models, and paves the way for further exploration of combined BMI1 and MAPK targeting in G4 MB patients.     

食管鳞癌ECA109细胞中Survivin通过ERK信号通路调控c-myc基因表达的机制

目的：探讨在食管鳞癌ECA109细胞中Survivin对c-myc基因的调控作用。方法：将食管鳞癌ECA109细胞分为Survivin shRNA干扰组、阴性质粒对照组（Control shRNA）和空白细胞株（未加任何处理的食管癌ECA109细胞）,将2μg Survivin shRNA质粒、2μg Control shRNA质粒分别转染ECA109细胞,48 h后收集各组细胞,采用RT-PCR法检测各组Survivin、c-myc mRNA的表达,Western blot法检测Survivin、c-myc蛋白及p-ERK蛋白的表达;采用ERK、p38、JNK、JAK/STA3、PI3K/Akt信号传导通路的特异性抑制剂分别阻断ECA109细胞中关键激酶的表达,RT-PCR法检测c-myc mRNA的表达。结果：与阴性质粒对照组和空白细胞组比较,Survivin shRNA组Survivin表达下调,c-myc mRNA及蛋白的表达降低,差异均具有统计学意义（P均 < 0.05）;采用ERK、p38、JNK、JAK/STA3、PI3K/Akt信号通路抑制剂分别作用于食管癌ECA109细胞,PD98059（ERK信号通路阻断剂）组c-myc mRNA及蛋白表达降低（P < 0.05）;Survivin shRNA干扰沉默Survivin基因后ERK磷酸化蛋白表达降低（P < 0.05）。结论：Survivin对c-myc具有正向调控作用,可能通过ERK信号传导通路调控c-myc的表达。     

Indirubin-3'-monoxime inhibits autophosphorylation of FGFR1 and stimulates ERK1/2 activity via p38 MAPK

Indirubin-3'-monoxime is a derivative of the bis-indole alkaloid indirubin, an active ingredient of a traditional Chinese medical preparation that exhibits anti-inflammatory and anti-leukemic activities. Indirubin-3'-monoxime is mainly recognized as an inhibitor of cyclin-dependent kinases (CDKs) and glycogen synthase kinase-3. It inhibits proliferation of cultured cells, mainly through arresting the cells in the G1/S or G2/M phase of the cell cycle. Here, we report that indirubin-3'-monoxime is able to inhibit proliferation of NIH/3T3 cells by specifically inhibiting autophosphorylation of fibroblast growth factor receptor 1 (FGFR1), blocking in this way the receptor-mediated cell signaling. Indirubin-3'-monoxime inhibits the activity of FGFR1 at a concentration lower than that required for inhibition of phosphorylation of CDK2 and retinoblastoma protein and cell proliferation stimulated by fetal calf serum. The ability of indirubin-3'-monoxime to inhibit FGFR1 signaling was similar to that of the FGFR1 inhibitor SU5402. In addition, we found that indirubin-3'-monoxime activates long-term p38 mitogen-activated protein kinase activity, which stimulates extracellular signal-regulated kinase 1/2 in a way unrelated to the activity of FGFR1. Furthermore, we show that indirubin-3'-monoxime can inhibit proliferation of the myeloid leukemia cell line KG-1a through inhibition of the activity of the FGFR1 tyrosine kinase. The data presented here demonstrate previously unknown activities of indirubin-3'-monoxime that may have clinical implications.     

TRPV4通过调控MAPK/ERK信号通路促进卵巢癌细胞的增殖和迁移北大核心

目的:探讨TRPV4通过影响MAPK/ERK信号通路调控卵巢癌细胞的增殖与迁移。方法:RT-qPCR和Western Blot方法检测TRPV4在卵巢癌细胞系和正常卵巢上皮细胞中的表达差异;GEPIA在线数据库分析卵巢癌患者TRPV4表达水平和预后关系;敲低TRPV4,通过RT-qPCR和Western Blot检测转染效率;通过细胞计数试剂盒(CCK8)、克隆形成实验以及Transwell迁移实验检测TRPV4在卵巢癌发生发展过程中增殖及迁移作用;通过GSEA软件富集分析信号通路并用Western Blot验证卵巢癌细胞MAPK/ERK信号通路表达量变化。结果:在卵巢癌细胞及临床组织样本中,TRPV4表达量明显增高,TRPV4表达量较高的患者预后更差。siRNA转染卵巢癌HO8910和Caov3细胞后,与Control组相比,TRPV4 mRNA和蛋白表达显著降低;细胞增殖迁移能力显著减弱;WB实验结果显示ERK总量不变,磷酸化水平降低,进一步实验中证实:敲低TRPV4后,ERK信号通路中的关键蛋白(P90RSK、MEK1/2、MSK1)磷酸化水平降低。结论:TRPV4在卵巢癌细胞及组织中高表达,可能通过正向调控MAPK/ERK信号通路,促进卵巢癌细胞增殖迁移等功能。