Targeted genomic CRISPR-Cas9 screen identifies MAP4K4 as essential for glioblastoma invasion
Laura M. ProloAmy LiScott F. OwenJonathon J. ParkerKara M. FoshayRyan T. NittaDavid W. MorgensSara BolinChristy WilsonJohana C. M. Vega LEmily LuoGigi NwagboAllen WaziriGordon LiRichard J. ReimerMichael C. BassikGerald A. Grant
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Abstract:
Among high-grade brain tumors, glioblastoma is particularly difficult to treat, in part due to its highly infiltrative nature which contributes to the malignant phenotype and high mortality in patients. In order to better understand the signaling pathways underlying glioblastoma invasion, we performed the first large-scale CRISPR-Cas9 loss of function screen specifically designed to identify genes that facilitate cell invasion. We tested 4,574 genes predicted to be involved in trafficking and motility. Using a transwell invasion assay, we discovered 33 genes essential for invasion. Of the 11 genes we selected for secondary testing using a wound healing assay, 6 demonstrated a significant decrease in migration. The strongest regulator of invasion was mitogen-activated protein kinase 4 (MAP4K4). Targeting of MAP4K4 with single guide RNAs or a MAP4K4 inhibitor reduced migration and invasion in vitro. This effect was consistent across three additional patient derived glioblastoma cell lines. Analysis of epithelial-mesenchymal transition markers in U138 cells with lack or inhibition of MAP4K4 demonstrated protein expression consistent with a non-invasive state. Importantly, MAP4K4 inhibition limited migration in a subset of human glioma organotypic slice cultures. Our results identify MAP4K4 as a novel potential therapeutic target to limit glioblastoma invasion.Keywords:
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Objective: To observe the G protein-coupled receptor 48 (GPCR48) expression in hepatocellular carcinoma (HCC) cell lines with different metastatic potential and its characteristics effect on the invasion and metastasis of Huh7 hepatoma cells via epithelial-mesenchymal transition (EMT). Methods: Western blot was used to detect the protein expression level of GPCR48 in HCC cells with different metastatic potential. The lentivirus vector expressing GPCR48 gene was constructed. GPCR48 was overexpressed in Huh7 hepatoma cells. The GPCR48 overexpression level was detected by real-time PCR and Western blot. Transwell invasion and migration assay was used to detect the Huh7 hepatoma cells invasion and migration ability in the Control, Mock and GPCR48 overexpression group. Real-time PCR and Western blot were used to detect Huh7 hepatoma cells mRNA and protein expression levels of the EMT related markers (E-cadherin, N-cadherin, vimentin, and γ catenin) in the Control, Mock and GPCR48 overexpression groups, respectively. Analysis of variance was used to compare the differences between data sets. Results: GPCR48 protein expression level in metastatic HCC cell lines was significantly higher than non-metastatic HCC cell lines (P < 0.05). The lentivirus vector expressing the GPCR48 gene had effectively transfected the Huh7 hepatoma cells and stably expressed the GPCR48mRNA and protein. Compared with the Mock and the Control group, Huh7 hepatoma cells invasion and migration ability in the GPCR48 overexpression group was significantly enhanced (F≥5.54, P < 0.05), and the mRNA and protein expression levels of epithelial phenotypic markers E-cadherin and γ-catenin were decreased (P < 0.05). The mRNA and protein expression levels of the mesenchymal phenotypic markers N-cadherin and Vimentin were increased (P < 0.05), indicating that EMT changes occurred in Huh7 hepatoma cells had overexpressed GPCR48. Conclusion: GPCR48 expression level is positively correlated with the metastatic potential of HCC cells. GPCR48 overexpression can down-regulate the expression of epithelial phenotypic markers and up-regulate the expression of mesenchymal phenotypic markers, and induce EMT changes in HCC cells, thus promoting HCC cells invasion and migration.目的： 观察G蛋白偶联受体48 (GPCR48)在不同转移潜能肝癌细胞株中的表达及其对肝癌细胞Huh7上皮间质转化(EMT)特性和侵袭转移的影响。 方法： 采用蛋白质印迹（Western blot）检测不同转移潜能肝癌细胞GPCR48的蛋白表达水平。构建携带GPCR48基因的慢病毒载体，在肝癌细胞Huh7过表达GPCR48；采用Real-time PCR和Western blot检测GPCR48的过表达水平，采用Transwell侵袭实验和迁移实验检测对照组、Mock组和GPCR48过表达组肝癌细胞Huh7的侵袭和迁移能力；采用Real-time PCR和Western blot检测对照组、Mock组和GPCR48过表达组肝癌细胞Huh7 EMT相关标志物E-钙黏蛋白（E-cadherin）、N-钙黏蛋白（N-cadherin）、波形蛋白（Vimentin）和γ连环蛋白（γ-catenin）的mRNA和蛋白的表达水平。数据组间差异比较采用方差分析。 结果： GPCR48蛋白表达水平在转移性肝癌细胞株明显高于非转移性肝癌细胞株（P < 0.05）。携带GPCR48基因的慢病毒载体可以有效转染肝癌细胞Huh7，稳定表达GPCR48的mRNA和蛋白。GPCR48过表达组肝癌细胞Huh7与Mock组和对照组相比，侵袭和迁移能力明显增强（F≥5.54，P值均< 0.05)，上皮表型标志物E-cadherin和γ-catenin的mRNA和蛋白表达水平下降（P值均<0.05)，间质表型标志物N-cadherin和Vimentin的mRNA和蛋白表达水平上升（P值均< 0.05），表明过表达GPCR48的肝癌细胞Huh7发生了EMT改变。 结论： GPCR48表达水平与肝癌细胞转移潜能呈正相关，过表达GPCR48可以下调上皮表型标志物的表达和上调间质表型标志物的表达，诱导肝癌细胞发生EMT改变，从而促进肝癌细胞侵袭和迁移。.
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Overexpression of Ebp1 inhibits the invasion and motility of adenoid cystic carcinoma cells in vitro
Objective To investigate the effect of Ebp1 overexpression on the invasion and motility of human adenoid cystic carcinoma(ACC) cells and its underlying mechanism.Methods Expression vector pcDNA3.1-Ebp1 was constructed,and cell lines stably expressed Ebp1 was established.Wound health assay and Transwell method were used to examine the invasion and motility of ACC cells.Matrix metalloprotease 9(MMP-9),intercelluar adhesion molecule 1(ICAM-1) and E-cadherin were detected by Western blot after Ebp1 overexpression.Results Numbers of cross-membrane cells in control,vector and Ebp1 groups were 33±7,92±11 and 106±5(P=0.03) in the invasion assay,and 68±5,168±2 and 203±4(16 fields,P=0.02) in the motility assay.Control and vector groups both took 16 hours for wound health,while the time in Ebp1 group was more than 24 hours(P=0.002).After Ebp1 overexpression,MMP-9 was downregulated,while ICAM-1 and E-cadherin were upregulated(P0.01).Conclusions Ebp1 inhibits the migration ability of ACC cells,which may be associated with decreasing of matrix degradation,enhancement of adhesion among cells and inhibition of epithelial mesenchymal transition.
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Adenoid Cystic Carcinoma
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Our previous study found that nicotine could induce lung cancer cell epithelial-mesenchymal transition (EMT). The aim of this study is to explore the relationship between nicotine-induced EMT and lung cancer invasion and metastasis.Real-time PCR and Western blot were used to detect the expression changes of EMT-related markers, E-cadherin and Vimentin, in A549 lung cancer cells treated with nicotine; The transposition of β-catenin protein expression was determined by immunofluorescence; Scratch test and Transwell invasion assay were used to detect the effects of nicotine on lung cancer cell migration and invasion.Nicotine can significantly down-regulate the expressional level of E-cadherin mRNA and protein of A549 cells in a manner of dose and time-dependent (P<0.01, P<0.01); Nicotine can significantly up-regulate the expressional level of Vimentin mRNA and protein of A549 cells in a manner of dose and time-dependent (P<0.01, P<0.01); Immunofluorescence results showed that β-catenin protein was significantly transfered to nucleus; Scratch test and Transwell assay showed that Nicotine could remarkably increase the migration and invasion potential of lung cancer cells (P<0.01, P<0.01).Nicotine can induce cancer cells EMT, and promote the invasion and metastasis ability of lung cancer cells.背景与目的 我们的前期研究发现尼古丁能诱导肺癌细胞上皮间质转化。本研究的目的是探讨尼古丁诱导的上皮间质转化（epithelial-mesenchymal transition, EMT）与肺癌侵袭之间的关系。方法 应用不同浓度尼古丁处理肺腺癌A549细胞，应用Real-time PCR和Western blot方法检测EMT相关分子标志物E-钙粘蛋白（E-cadherin）和波形蛋白（Vimentin）mRNA和蛋白表达水平，应用免疫荧光技术检测β-链蛋白（β-catenin）蛋白表达位置的变化，应用划痕实验和Transwell小室侵袭实验检测尼古丁对肺癌细胞迁移侵袭能力的影响。结果 尼古丁明显下调肺癌细胞株A549 E-cadherin mRNA和蛋白水平表达（P<0.01, P<0.01），并具有浓度和时间依赖性；尼古丁明显上调肺癌细胞株A549 Vimentin mRNA和蛋白水平表达（P<0.01, P<0.01）；尼古丁诱导肺癌细胞株A549细胞β-catenin蛋白发生核转移；划痕实验和侵袭实验观察到尼古丁处理的肺癌细胞株A549细胞的迁移和侵袭能力明显增强（P<0.01, P<0.01）。结论 尼古丁能够诱导肺癌细胞发生EMT，并且促进肺癌细胞株A549细胞的体外侵袭潜能。.
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Directional motility and invasion assays are largely based on the use of Boyden chambers or Transwell™ culture inserts in which porous membranes separate seeded cells from a chemotactic factor supplied in the medium outside the chamber. The major obstacle for most currently available assays is that they lack a sensitive, easy, and reliable method of quantifying the nonmotile cell populations. Failure to account for all cells within the assay chamber prohibits the determination of percentages of migrated cells. Here we describe an ATP luminescence-based motility-invasion (ALMI) assay that circumvents this problem, enabling investigators to quantify directional cell migration or invasiveness easily. The ALMI assay is based on the detection of ATP in viable cells harvested from inert surfaces that do not generate background signals. We demonstrate how the ALMI assay can be used to assess the effects of various experimental conditions such as growth factor stimulation and ethanol exposure on cell migration. In addition, precoating the membranes with extracellular matrix molecules enabled the measurement of the cell invasion. In conclusion, the ALMI assay provides a reliable and flexible method to quantify cell motility and invasiveness using a luminescence microplate reader.
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Squamous carcinoma
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Objective To evaluate the effects of resveratrol(Rev) on migration and invasion of glioma cell line U87 cells and its mechanism.Methods Viability of glioma U87 cells treated with Rev in variable concentrations for 24 h and 48 h was detected by methyl thiazolyl tetrazolium(MTT) assay.The migration and invasion were evaluated by in vitro scratch assay and Boyden chamber assay respectively;matrix-metalloproteinase(MMP) activity was determined by gelatine zymography assay.Results MTT assay showed Rev inhibited the proliferation of glioma U87 cells in a dose-and time-dependent manner(P0.05);compared with control group,the migration and invasion of U87 cells treated with Rev were decreased obviously,accompanied by the inhibition of MMP-2 activity.Conclusion Rev can inhibit the migration and invasion ability of glioma U87 cells.The inhibitive effects may be associated with the decreased MMP-2 activity.
MTT assay
U87
Viability assay
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Matrix metalloproteinase 9
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AIM: Glioblastoma multiforme (GBM) is the most common primary brain tumor in humans, and it is highly invasive. Doxycycline, first identified as an antimicrobial agent, is a nonspecific inhibitor of matrix metalloproteinases (MMPs). Our objective was to investigate the anti-MMP effect of doxycycline at therapeutically acceptable levels on glioma cells in vitro. METHODS: The MTT assay was used to determine the anti-proliferative effects of doxycycline. MMP2 activity and expression were determined by gelatinase zymography and real-time quantitative RT-PCR, respectively. Cell invasion was assessed by Matrigel invasion assay. RESULTS: Doxycycline exerted mild anti-proliferative effects on all three glioma cell lines (U251HF, U87 and LN229). In U251HF cells, doxycycline decreased extracellular MMP2 activity and reduced cell invasiveness. Moreover, MMP2 mRNA levels were not altered, suggesting that doxycycline regulates MMP2 activity post-translationally. Alternatively, doxycycline increased the expression and extracellular activity of MMP2 in U87 cells. This may reflect the cellular stress-response related to the cytotoxic effects experienced by U87 cells in response to doxycycline exposure. CONCLUSION: Doxycycline in therapeutic concentrations decreases MMP2 activity and cell invasion in the most aggressive cell line tested, suggesting its potential as a therapeutic MMP inhibitor. The cytotoxic effects of doxycycline, however, can enhance MMP2 expression, and this deserves further exploration.
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U87
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