A正向遗传筛选标识肝细胞癌发生的抑制器

StephenW

A Forward Genetic Screen Identifies Suppressors of Hepatocellular Carcinogenesis

   
8:46 PM (2 hours ago)
        

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A Forward Genetic Screen Identifies Suppressors of Hepatocellular Carcinogenesis
by Kristine Novak

Using 2 powerful high-throughput in vivo screening tools, researchers identified and validated 27 genes as suppressors of hepatocellular carcinoma (HCC), reported in the August issue of Gastroenterology. Their findings support the role of Ras signaling in development of HCC and provide new therapeutic targets.

Sorafenib is the only treatment for patients with advanced-stage HCC, but this drug prolongs patient survival by only a few months. To discover effective treatments for HCC we need to learn more about the mechanisms of HCC development.

Genetic analyses have identified only a few genes (TERT, CTNNB1, TP53, and ARID2) that are mutated in more than 5% of HCCs. In contrast, infrequent mutations (<5% of patients) have been identified in more than 10,000 genes. HCC is therefore a highly heterogenous tumor.

The 2-step forward genetic screen in mice using the sleeping beauty transposon system and shRNA library

The 2-step forward genetic screen in mice using the sleeping beauty transposon system and shRNA library

In contrast to these reverse genetic analyses, which start with a biologic feature and look for gene variants that cause it, in forward genetic analyses, researchers disrupt random genetic sequences and see what phenotypes develop. This approach provides an unbiased selection for genes that control a phenotype of interest, such as HCC development.

A forward genetic screen using sleeping beauty transposon mutagenesis of transgenic mice predisposed to develop hepatocellular adenoma and HCC (as a result of the expression of hepatitis B surface antigen, HBsAg) had previously identified 21 genes that regulated liver tumorigenesis and tumor progression. In this system, cells that acquire a survival advantage, through transposon-mediated activation or inactivation, give rise to tumors. However, this original system could not cover the entire genome, due to technical limitations.

Takahiro Kodama et al used a system (T2Onc3) that overcame these obstacles and increased the number of tumor-associated genes identified by sleeping beauty-based mutagenesis. They then screened a high-throughput short hairpin RNA (shRNA) library for the effects of knockdown of these genes in immortalized mouse liver cells.

Mice that carried the HBsAg transgene and were undergoing active T2Onc3 transposition in their liver started to develop tumors at approximately 1 year of age, developed more advanced tumors, and had significantly shorter survival times than HBsAg mice without active sleeping beauty transposition. Even some mice with the transposon without HBsAg developed advanced liver tumors.

Kodama et al identified 1917 genes at which transposon insertion promoted liver tumor development. Analyses of their products revealed the importance of Ras signaling in the development of HCC.

A shRNA library screen of 250 selected genes validated 27 suppressors of HCC development. Individual shRNA knockdown of 4 of these tumor suppressor genes (Acaa2, Hbs1l, Ralgapa2, and Ubr2) increased proliferation of multiple human HCC cell lines and accelerated formation of xenograft tumors in nude mice. The authors concluded that these genes are bona fide tumor suppressors in mouse and human liver.

When the authors compared the outcomes of patients whose HCCs carried a mutation in these tumor suppressors, using a publically available microarray data set, they found that expression levels of 9 of these genes had a negative correlation with patient survival.

Ralgapa2 encodes a member of the Ral guanosine triphosphatase (GTPase)-activating protein (RalGAP) family, which suppresses the oncogenic Ral pathway. The authors found that 51% of sleeping beauty-induced tumors had a transposon insertion in 1 or more of these family members.

The ability of Ralgapa2 knockdown to promote HCC cell proliferation and tumor formation required expression of Rala and Ralb. Dual inhibition of Ras signaling via RAL and RAF, using a combination of small-molecule inhibitor RBC8 and sorafenib, reduced the proliferation of HCC cells in culture and completely inhibited their growth as xenograft tumors in nude mice.

Ralgapa2, and the closely related Ralgapa1 and Ralgapb family members, negatively regulate the RalA and RalB Ras-like small GTPases through the hydrolysis of GTP. Although oncogenic roles for the Ral pathway have been reported in pancreas, skin, lung, colon, prostate, and bladder cancer, little is known about the mechanisms of RalGAPs in cancer development. Down-regulation of RALGAPA2 promotes invasion and metastasis of bladder cancer. Kodama et al showed that that RalGAPs suppress cell proliferation and growth of xenograft tumors from HCC cells via inactivation of the Ral pathway.

Kodama et al noted that the oncoding RNA Rian was mutated in the highest number of tumors and showed a dense pattern of insertions in the antisense strand. Sleeping beauty insertions at Rian promoted liver tumor development through activation of the promoter of an adjacent oncogene, Rtl, rather than through insertional inactivation of Rian.

The second and third most highly ranked trunk drivers (genes mutated or deregulated at an early stage of tumor development) identified were Hras and Kras. Although Ras is not frequently mutated in HCCs, this pathway is activated in 50%–100% of HCCs, through down-regulation of Ras inhibitors such as GTPase-activating proteins and Rassf and Spred family members. So the Ras pathway appears to be a good target for development of anti-HCC agents. Sorafenib targets RAF/MEK/ERK members of the Ras pathway.

In an editorial that accompanies the article, Andreas Teufel and Jean François Dufour write that the findings nicely demonstrate the usefulness of the sleeping beauty transposon system, in combination with simultaneous gene expression profile analyses, to identify genes that regulate tumor development. The findings could provide new therapeutic targets for HCC and support the investigation of transposon systems for cancer gene therapy.

Kristine Novak | August 23, 2016 at 6:46 am | Tags: mice, Ralgapa, Ras, transposable element, transposon | URL: http://wp.me/p4B9rV-1GY   

StephenW

A正向遗传筛选标识肝细胞癌发生的抑制器


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A正向遗传筛选标识肝细胞癌发生的抑制器
由克里斯汀·诺瓦克

使用2强大的高通量体内筛选工具，研究人员鉴定和验证27基因为肝细胞癌（HCC）的抑制，报道胃肠病八月问题。他们的发现支持Ras信号的作用，肝癌的发展提供新的治疗靶点。

索拉非尼是治疗晚期肝癌的唯一治疗，但这种药物只有几个月延长患者生存期。要发现有效的治疗肝癌，我们需要更多地了解肝癌的发展机制。

遗传分析已经确定只有少数基因（TERT，CTNNB1，TP53和ARID2），它们在肝癌的5％以上的突变。相比之下，罕见突变（<5％的患者）已经在超过10000个基因鉴定。因此，肝癌是一种高度异质性肿瘤。

在两级正向遗传学屏幕使用睡美人转座子体系和shRNA文库小鼠

在两级正向遗传学屏幕使用睡美人转座子体系和shRNA文库小鼠

相对于这些反向遗传分析，其与生物特征就寻找基因的变体导致它，在向前遗传分析，研究人员扰乱随机基因序列和看什么表型发展。这种方法提供了用于控制感兴趣的表型的基因，如肝癌发展一个无偏的选择。

利用转基因小鼠的睡美人转座子诱变正向遗传筛选倾向于发展肝细胞腺瘤和HCC（如B型肝炎表面抗原的表达的结果）先前确定了调节肝脏肿瘤发生和肿瘤进展21基因。在这个系统中，获得存活优势，通过转座子介导的激活或失活的细胞，引起肿瘤。然而，这种原有的系统无法覆盖整个基因组中，由于技术的限制。

宙Kodama等人使用了克服这些障碍，并增加由睡美人基于诱变鉴定的肿瘤相关基因的数量的系统（T2Onc3）。然后，他们筛选高吞吐量短发夹RNA（shRNA）库这些基因在永生化小鼠肝细胞的敲低效果。

小鼠携带的HBsAg的转基因和正在接受活性T2Onc3换位在其肝脏开始发展的肿瘤在约1岁，开发更先进的肿瘤，并且具有比HBsAg的小鼠存活时间显著短无活动睡美人换位。即使是一些小鼠没有的HBsAg转座子开发了先进的肝肿瘤。

儿玉等人确定了1917个基因在其中转座子插入促进肝肿瘤的发展。他们的产品的分析显示在HCC的发展Ras信号的重要性。

250选定的基因一shRNA文库屏幕验证HCC发展27抑制器。增加的多种人类肝癌细胞株增殖和裸鼠加速形成移植瘤，这些肿瘤抑制基因（ACAA2，HBS1L，Ralgapa2和Ubr2）的4个独立的shRNA击倒。作者认为，这些基因在小鼠和人类肝脏善意的肿瘤抑制基因。

当作者比较的患者的肝细胞癌在这些肿瘤抑制携带的突变，使用公开可用的微阵列数据集的结果，他们发现，这些基因的9表达水平与患者存活的负相关性。

Ralgapa2编码拉尔鸟苷三磷酸（GTP酶）-activati​​ng蛋白（RalGAP）家族，其抑制致癌拉尔途径的成员。作者发现，睡美人诱导肿瘤的51％，以1或多个家庭成员有一个转座子插入。

Ralgapa2击倒，促进肝癌细胞增殖和肿瘤形成的能力要求和拉拉的Ralb表达。的的Ras经由RAL和RAF信令，使用小分子抑制剂RBC8和索拉非尼的组合双重抑制，减少的肝癌细胞的增殖培养和完全抑制其生长在裸鼠异种移植肿瘤。

Ralgapa2，和紧密相关的Ralgapa1和Ralgapb家庭成员的，带负通过GTP的水解·拉拉和RalB的Ras样小GTP酶调节。尽管对于拉尔通路致癌作用已在胰腺癌，皮肤癌，肺癌，结肠癌，前列腺癌和膀胱癌的报道，知之甚少RalGAPs在癌症发展的机制。下调RALGAPA2的促进侵袭和膀胱癌的转移。儿玉等人发现，该RalGAPs通过拉尔途径的失活抑制细胞的增殖和肝癌细胞移植瘤的生长。

Kodama等人指出，oncoding RNA日安在肿瘤的最高数目突变并显示插入的反义链密集图案。在日安睡美人插入通过相邻的癌基因，RTL，而不是通过日安的插入失活的启动子的激活促进了肝脏肿瘤的发展。

确定的第二和第三个排名最高的主干驱动程序（基因突变或失调在肿瘤发展的早期阶段）为HRA和嘉仕。尽管拉斯不经常在肝癌发生突变，这一途径在50％的肝癌激活-100％，通过的Ras抑制剂下调，如GTP酶激活蛋白和RASSF和Spred家族成员。所以Ras通路似乎是抗HCC试剂的发展良好的目标。索拉非尼目标的Ras通路的RAF / MEK / ERK成员。

在附带的文章一篇社论，安德烈亚斯Teufel和Jean弗朗索瓦·杜福尔写道，研究结果表明很好的睡美人转座系统并同时基因表达谱的实用性，结合分析，以确定调控肿瘤发育的基因。这一发现可能为肝癌提供了新的治疗靶点和支持转系统的肿瘤基因治疗的研究。

克里斯汀·诺瓦克| 2016年8月23日在上午6时46 |标签：鼠标，Ralgapa，RAS，转座子，转座子|网址：http://wp.me/p4B9rV-1GY