A mammosphere formation RNAi screen reveals that ATG4A promotes a breast cancer stem-like phenotype.
A mammosphere formation RNAi screen reveals that ATG4A promotes a breast cancer stem-like phenotype.
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DOI：
10.1186/bcr3576
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发表时间：
2013-11-14
期刊：
影响因子：
--
通讯作者：
Boettcher M
中科院分区：
文献类型：
--
作者：
Wolf J;Dewi DL;Fredebohm J;Müller-Decker K;Flechtenmacher C;Hoheisel JD;Boettcher M
乳腺癌干细胞被怀疑与肿瘤复发、转移形成以及化疗耐药有关。因此，人们在了解癌症干细胞维持的分子机制方面做出了巨大的努力。为了在体外研究这些稀有细胞，它们通常通过乳腺球培养进行富集。在这里，我们开发了一种基于乳腺球的负选择shRNAi筛选系统，适用于分析数千个基因在具有癌症干细胞特性的细胞存活中的参与。我们描述了在SUM149中表达干细胞样标记CD44+/CD24-/low的亚群，这些亚群在乳腺球体中富集。为了确定在功能上参与维持具有癌症干细胞特性的亚群的基因，我们通过RNAi瞄准了5000多个基因，并测试了它们作为乳房微球生长的能力。鉴定的候选ATG4A在乳腺球和软琼脂集落形成实验中得到验证。进一步，我们评估了ATG4A表达对表达茎样标志物CD44+/CD24low的亚群的影响。接下来，通过异种移植实验检测上调或下调ATG4A后SUM149的致瘤潜能。通过这种方法，Jak-STAT和细胞因子信号被确定参与乳腺球的形成。此外，自噬调节因子ATG4A被发现对于维持具有癌症干细胞特性的亚群和调节体内乳腺癌细胞的致瘤性至关重要。总之，我们提出了一种高通量筛选系统来识别参与癌症干细胞维持的基因，并通过ATG4A证明了其实用性。
Breast cancer stem cells are suspected to be responsible for tumour recurrence, metastasis formation as well as chemoresistance. Consequently, great efforts have been made to understand the molecular mechanisms underlying cancer stem cell maintenance. In order to study these rare cells in-vitro, they are typically enriched via mammosphere culture. Here we developed a mammosphere-based negative selection shRNAi screening system suitable to analyse the involvement of thousands of genes in the survival of cells with cancer stem cell properties. We describe a sub-population expressing the stem-like marker CD44+/CD24-/low in SUM149 that were enriched in mammospheres. To identify genes functionally involved in the maintenance of the sub-population with cancer stem cell properties, we targeted over 5000 genes by RNAi and tested their ability to grow as mammospheres. The identified candidate ATG4A was validated in mammosphere and soft agar colony formation assays. Further, we evaluated the influence of ATG4A expression on the sub-population expressing the stem-like marker CD44+/CD24low. Next, the tumorigenic potential of SUM149 after up- or down-regulation of ATG4A was examined by xenograft experiments. Using this method, Jak-STAT as well as cytokine signalling were identified to be involved in mammosphere formation. Furthermore, the autophagy regulator ATG4A was found to be essential for the maintenance of a sub-population with cancer stem cell properties and to regulate breast cancer cell tumourigenicity in vivo. In summary, we present a high-throughput screening system to identify genes involved in cancer stem cell maintenance and demonstrate its utility by means of ATG4A.
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