Name: identification of egfr and ras inhibitors using caenorhabditis elegans
Uploaded: 2020-10-05T14:00:00
Description: Watch this Scientific Journal Video about Identification of EGFR and RAS Inhibitors using Caenorhabditis elegans at JoVE.com

我们感谢斯瓦蒂·阿鲁尔博士（MD安德森癌症中心）提供了 让60（n1046）。我们还要感谢大卫·雷纳博士（休斯顿得克萨斯A&M健康科学中心生物科学与技术研究所）的 林-1 菌株。最后，我们感谢丹妮尔·加辛博士和她的实验室（德克萨斯大学麦戈文医学院）提供了一些试剂。一些蠕虫菌株由国家卫生研究院基础设施项目办公室（P40 OD010440）资助的CGC提供。这项研究得到了德克萨斯州癌症预防和研究所（CPRIT）向JF汉考克提供RP200047的支持。

1. 线虫生长介质 （NGM） 板准备
<ol><li>在 2 L 埃伦迈尔烧瓶中加入 2.5 克辣椒和 3 克 NaCl 到 970 mL 的去离子水。使用磁搅拌棒搅拌内容。此后，在烧瓶中加入20克醋。在 121 °C 和 15 磅/在2 的压力下将烧瓶的含量高压 30 分钟。消毒后，将烧瓶放在搅拌盘上，让中等温度冷却至 50 °C。</li>
	<li>要准备 NGM 板，请在冷却介质中添加以下试剂：25 mL 的 1 M 磷酸钾缓冲器 （pH = 6.0），1 mL 的 1 M MgSO4...

<ol>
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我们描述使用蠕虫的检测是简单和廉价的，以确定EGFR和RAS功能的抑制剂。 C. elegans 是药物发现有吸引力的模型，因为由于生命周期短（3 天在 20 °C）和产生大量幼虫的能力，在实验室中很容易生长。更重要的是，EGFR-RAS-ERK MAPK通路在进化和功能上得到保护，哺乳动物提供了一个基因可传导系统来分析EGFR和RAS抑制剂的影响。此外，蠕虫的透明性使研究者能够可视化不同的结构，以及绿色荧光...

调节生物体内发育事件的细胞通路在所有元子中都得到了高度保护。其中一个途径是EGFR-RAS-ERK线粒体活性蛋白激酶（MAPK）信号级联，这是控制细胞增殖、分化、迁移和生存的关键途径1，2。此信号通路中的缺陷可能导致病理或疾病状态，如癌症。表皮生长因子受体（EGFR）在人类肿瘤中表现出高度表达，包括50%的口腔鳞状细胞癌，并有助于恶性肿瘤3、4、5的发展。而三个RAS同位素H-，K-和N-RAS的突变是多种人类癌症恶性转化的主要驱动因素。在这三种RAS同位素中，K-RAS的致癌突变最为普遍，为6、7、8。要使EGFR和RAS发挥作用，它们必须定位到等离子膜（PM）。防止这些分子的本地化到PM可以完全废除这个信号通路9，10的生物活性。因此，抑制这些蛋白质的本地化到PM是一种治疗策略，以阻止下游信号和由此产生的不良后果。使用高含量筛选检测，芬迪林，L型钙通道阻滞剂，被确定为K-RAS活性的抑制剂11。在芬迪林的存在下，K-RAS 到 PM 内部传单的纳米集群显著减少。此外，K-RAS从等离子体膜重新分配到内质视网膜（ER）、高尔吉仪器、内分泌体和细胞溶胶。更重要的是，胰腺癌、结肠癌、肺癌和子宫内膜癌细胞系的增殖被芬迪林11抑制下游信号所阻断。这些数据表明，芬迪林作为一种特定的K-RAS抗癌疗法，导致RAS蛋白对PM的误定位。
线虫 卡诺哈布德炎埃莱甘斯 已在发展的背景下进行了广泛的研究。许多控制蠕虫发展的信号通路都是进化的，在功能上是保守的。例如，EGFR 介解 RAS 激活和随后激活的 ERK MAPK 信号级联在蠕虫12中保存。级联由以下蛋白质表示：LET-23 > LET-60 > LIN-45 > MEK-2 >MPK-1。LET-60 与 RAS 同源，而 LET-23 与 EGFR 同源。在蠕虫中，这种途径调节外阴13的发育。外阴是蠕虫腹壁上的上皮孔径，允许产卵。蠕虫外阴的形成取决于外阴前体细胞 （VPC） 暴露在 EGFR-RAS-MAPK 信号级联激活梯度下。在正常发育过程中，近亲VC从淋瑙锚细胞接收强信号，以分化成1°和2°细胞的命运，从而产生功能性外阴12。而离散性VC区分成3°细胞的命运，融合到皮下同步，不会形成外阴由于耗尽的信号。在没有信号的情况下，所有 VPC 都区分为 3° 细胞命运，从而形成无外阴。然而，构成信号导致形成一个或多个非功能性外阴，由于所有VPC的诱导，以承担1°和2°细胞的命运。
导致外阴诱导缺陷或过度的突变已经为代表该通路的蛋白质编码的许多基因被识别出来。有缺陷的外阴诱导导致无外阴（Vul）表型，而过度的外阴诱导导致多外阴（Muv）表型，其表现是在整个心室壁上出现大量非功能性异位伪外阴。让-60（n1046）菌株表达的Muv表型是由于RAS功能突变的增加，而在让-23（sa62）菌株中，它是由于EGFR14，15的激活突变。这些突变菌株中的强Muv表型已被药理干预所困扰，通过MEK-1抑制剂U0126 16、17治疗让-60（n1046）蠕虫就证明了这一点。有趣的是，我们已经表明，R-芬迪林和抑制剂，影响苯丙胺代谢抑制在蠕虫18的Muv表型。为了证明这些抑制剂块让60信号在RAS的水平，林-1空菌株已被使用17。Lin-1是一种类似Ets的抑制转录因子，在外阴19的发育中起到抑制作用。在让-60（n1046）蠕虫中，Muv表型的强烈回归，对lin-1空蠕虫没有影响，这表明这些抑制发生在RAS的水平。
在此协议中，我们演示了使用C. elegans作为模型来识别 RAS 和 EGFR 蛋白的抑制剂。使用液体基检测，我们通过抑制让-60（n1046）和让-23（sa62）突变菌株的C.elegans的Muv表型来证明R-芬迪林的抑制作用。这种检测验证了在抗癌治疗药物发现初始阶段使用C.elegans作为工具。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Media and chemicals</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Agarose&#160;</td>
			<td>Millipore Sigma&#160;</td>
			<td>A9539-50G</td>
			<td></td>
		</tr>
		<tr>
			<td>Bacto Peptone&#160;</td>
			<td>Fisher Scientific</td>
			<td>DF0118-17-0</td>
			<td></td>
		</tr>
		<tr>
			<td>BD Difco Agar&#160;</td>
			<td>Fisher Scientific</td>
			<td>DF0145-17-0</td>
			<td></td>
		</tr>
		<tr>
			<td>BD Difco LB Broth</td>
			<td>Fisher Scientific</td>
			<td>DF0446-17-3</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium Chloride</td>
			<td>Fisher Scientific</td>
			<td>BP510-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Cholesterol</td>
			<td>Fisher Scientific</td>
			<td>ICN10138201</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium Sulfate</td>
			<td>Fisher Scientific</td>
			<td>BP213-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Nystatin</td>
			<td>Acros organics</td>
			<td>AC455500050</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium Phosphate Dibasic</td>
			<td>Fisher Scientific</td>
			<td>BP363-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium pPhosphate Monobasic</td>
			<td>Fisher Scientific</td>
			<td>BP362-500</td>
			<td></td>
		</tr>
		<tr>
			<td>R-Fendiline</td>
			<td></td>
			<td></td>
			<td>Commercially Synthesized (Pharmaceutical grade)</td>
		</tr>
		<tr>
			<td>Sodium Azide</td>
			<td>Millipore Sigma&#160;</td>
			<td>S2002-25G</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride&#160;</td>
			<td>Fisher Scientific</td>
			<td>BP358-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Hydroxide</td>
			<td>Fisher Scientific</td>
			<td>SS266-1</td>
			<td></td>
		</tr>
		<tr>
			<td>8.25% Sodium Hypochlorite&#160;</td>
			<td></td>
			<td></td>
			<td>Bleach</td>
		</tr>
		<tr>
			<td>Sodium Phosphate Dibasic&#160;</td>
			<td>Fisher Scientific</td>
			<td>BP332-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Streptomycin Sulfate&#160;</td>
			<td>Fisher Scientific</td>
			<td>BP910-50</td>
			<td></td>
		</tr>
		<tr>
			<td>(&#8722;)-Tetramisole Hydrochloride</td>
			<td>Millipore Sigma&#160;</td>
			<td>L9756</td>
			<td></td>
		</tr>
		<tr>
			<td>UO126 (MEK inhibitor)</td>
			<td>Millipore Sigma&#160;</td>
			<td>19-147</td>
			<td></td>
		</tr>
		<tr>
			<td>Consumables&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>15mL Conical Sterile Polypropylene Centrifuge Tubes&#160;</td>
			<td>Fisher Scientific</td>
			<td>12-565-269</td>
			<td></td>
		</tr>
		<tr>
			<td>50mL Conical Sterile Polypropylene Centrifuge Tubes</td>
			<td>Fisher Scientific</td>
			<td>12-565-271</td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable Polystyrene Serological Pipettes 10mL</td>
			<td>Fisher Scientific</td>
			<td>07-200-574</td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable Polystyrene Serological Pipettes 25mL</td>
			<td>Fisher Scientific</td>
			<td>07-200-575</td>
			<td></td>
		</tr>
		<tr>
			<td>No. 1.5&#160; 18 mm X 18 mm Cover Slips</td>
			<td>Fisher Scientific</td>
			<td>12-541A</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri Dish with Clear Lid (60 x 15 mm)</td>
			<td>Fisher Scientific</td>
			<td>FB0875713A</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri Dishes with Clear Lid (100X15mm)</td>
			<td>Fisher Scientific</td>
			<td>FB0875712</td>
			<td></td>
		</tr>
		<tr>
			<td>Plain Glass Microscope Slides (75 x 25 mm)</td>
			<td>Fisher Scientific</td>
			<td>12-544-4</td>
			<td></td>
		</tr>
		<tr>
			<td>12- Well Tissue Culture Plates</td>
			<td>Fisher Scientific</td>
			<td>50-197-4804</td>
			<td></td>
		</tr>
		<tr>
			<td>Software&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Prism</td>
			<td>Graphpad</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Bacterial Strains</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>E. coli OP50</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Worm Strains</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Strain</td>
			<td>Genotype</td>
			<td>Transgene</td>
			<td>Source</td>
		</tr>
		<tr>
			<td>MT2124 &#160;</td>
			<td>let-60(n1046) IV.</td>
			<td></td>
			<td>CGC</td>
		</tr>
		<tr>
			<td>MT7567</td>
			<td>lin-1(sy254) IV.</td>
			<td></td>
			<td>CGC</td>
		</tr>
		<tr>
			<td>PS1839</td>
			<td>let-23(sa62) II.</td>
			<td></td>
			<td>CGC</td>
		</tr>
	</tbody>
</table>

identification of egfr and ras inhibitors using caenorhabditis elegans

表皮生长因子受体（EGFR）及其下游效应器RAS的血浆膜定位变化与包括癌症在内的多种疾病有关。自由活的线虫 C.elegans 具有进化和功能保存的EGFR-RAS-ERK MAP信号级联，是外阴发育的核心。RAS同源LET-60和EGFR同源LET-23功能突变的增益诱导这些蠕虫的腹壁产生可见的非功能性异位伪外阴。以前，这些蠕虫中的多外阴（Muv）表型已被小化学分子抑制。在这里，我们描述了在液体基检测中使用蠕虫来识别消除EGFR和RAS蛋白活动的抑制剂的协议。通过此检测，我们显示 K-RAS 的间接抑制剂 R-fendiline 抑制 了在 let-60 （n1046） 和 让-23 （sa62） 突变蠕虫中表达的 Muv 表型。检测简单、价格低廉，设置不费时，可作为发现抗癌治疗的初始平台。

我们首先证明，与DMSO治疗的蠕虫相比，R-fendiline能够抑制 让-60（n1046） 突变菌株中的Muv表型。我们的数据显示，R-芬迪林能够以依赖剂量的方式阻断 让-60（n1046） 中的Muv表型（图2A，B）。然而，在 林-1 空突变菌株中观察到Muv表型的未逆转，以响应R-芬迪林浓度的增加（图2B）。数据表明，R-芬迪林块激活 让60 信?...

Watch this Scientific Journal Video about Identification of EGFR and RAS Inhibitors using Caenorhabditis elegans at JoVE.com

Identification of EGFR and RAS Inhibitors using Caenorhabditis elegans

基因可处理的线虫 卡诺哈布迪炎的叶原体 可以用作药物发现一个简单和廉价的模型。这里描述的是一个协议，以确定抗癌治疗，抑制RAS和EGFR蛋白的下游信号。

使用卡诺哈布迪炎埃莱甘斯识别 EGFR 和 RAS 抑制剂

The changes in the plasma membrane localization of the epidermal growth factor receptor (EGFR) and its downstream effector RAS have been implicated in ...

Research

JoVE Journal

Cancer Research

Identification of EGFR and RAS Inhibitors using Caenorhabditis elegans

Sectioning Mammary Gland Whole Mounts for Lesion Identification

切片乳腺全身为病变鉴别

Normal mammary gland development may be altered by exposure to environmental toxicants and pharmaceutical products, excessive exposure to hormones, and ...

科研

癌症研究

Identification and Characterization of Metastatic Factors by Gene Transfer into the Novel RIP-Tag; RIP-tva Murine Model

Identification and Characterization of Metastatic Factors by Gene Transfer into the Novel RIP-Tag; RIP-tva Murine Model

转移因子的鉴定和表征转移到新的RIP 标签;tva小鼠模型

Metastatic cancer accounts for 90% of deaths in patients with solid tumors. There is an urgent need to better understand the drivers of cancer metastasis ...

Establishing Dual Resistance to EGFR-TKI and MET-TKI in Lung Adenocarcinoma Cells In Vitro with a 2-step Dose-escalation Procedure

Establishing Dual Resistance to EGFR-TKI and MET-TKI in Lung Adenocarcinoma Cells In Vitro with a 2-step Dose-escalation Procedure

建立双抗 EGFR-使用 tki 药物治疗和满足使用 tki 药物治疗在肺腺癌细胞体外与 2 步剂量升级程序

Drug resistance is a major challenge in cancer therapy. The generation of resistant sublines in vitro is necessary for discovering novel mechanisms to ...

Identification of OTX1 and OTX2 As Two Possible Molecular Markers for Sinonasal Carcinomas and Olfactory Neuroblastomas

otx1 和 otx2 作为鼻窦癌和嗅觉神经母细胞瘤的两种可能的分子标记物的鉴定

OTX homeobox (HB) genes are expressed during embryonic morphogenesis and during the development of olfactory epithelium in adult organisms. Mutations ...

Assessment of Resistance to Tyrosine Kinase Inhibitors by an Interrogation of Signal Transduction Pathways by Antibody Arrays

用抗体阵列对信号转导通路的审问对酪氨酸激酶抑制剂的抗性评价

Cancer patients with an aberrant regulation of the protein phosphorylation networks are often treated with the tyrosine kinase inhibitors. Response rates ...

Identification, Histological Characterization, and Dissection of Mouse Prostate Lobes for In Vitro 3D Spheroid Culture Models

体外3D 球形培养模型小鼠前列腺叶的鉴定、组织学特征及解剖

Genetically engineered mouse models (GEMMs) serve as effective pre-clinical models for investigating most types of human cancers, including prostate ...

Characterization of the Effects of Migrastatic Inhibitors on 3D Tumor Spheroid Invasion by High-resolution Confocal Microscopy

通过高分辨率共聚焦显微镜对3D肿瘤球体入侵的影响表征

Drug discovery and development in cancer research is increasingly being based on drug screens in a 3D format. Novel inhibitors targeting the migratory and ...

Identification of Transcription Factor Regulators using Medium-Throughput Screening of Arrayed Libraries and a Dual-Luciferase-Based Reporter

使用阵列库的中等通量筛选和基于双 Luciferase 的分录报告器识别转录因子稳压器

Transcription factors can alter the expression of numerous target genes that influence a variety of downstream processes making them good targets for ...

Assays for Validating Histone Acetyltransferase Inhibitors

验证组蛋白乙酰转移酶抑制剂的检测

Lysine acetyltransferases (KATs) catalyze acetylation of lysine residues on histones and other proteins to regulate chromatin dynamics and gene ...

Assessing Cellular Target Engagement by SHP2 (PTPN11) Phosphatase Inhibitors

Assessing Cellular Target Engagement by SHP2 PTPN11 Phosphatase Inhibitors

通过 SHP2 （PTPN11） 磷酸酶抑制剂评估细胞目标参与度

The Src-homology 2 (SH2) domain-containing phosphatase 2 (SHP2), encoded by the PTPN11 proto-oncogene, is a key mediator of receptor tyrosine kinase ...