Cancer Research Program, Houston Methodist Research Institute, Houston, Texas.
Institute of Molecular and Cell Biology, Agency for Science, Technology and Research, Biopolis, Singapore.
Gastroenterology. 2016 Aug;151(2):324-337.e12. doi: 10.1053/j.gastro.2016.04.040. Epub 2016 May 10.
BACKGROUND & AIMS: High-throughput sequencing technologies have identified thousands of infrequently mutated genes in hepatocellular carcinomas (HCCs). However, high intratumor and intertumor heterogeneity, combined with large numbers of passenger mutations, have made it difficult to identify driver mutations that contribute to the development of HCC. We combined transposon mutagenesis with a high-throughput screen of a small-hairpin RNA (shRNA) library to identify genes and pathways that contribute to HCC development.
Sleeping beauty transposons were mobilized in livers of transgenic mice predisposed to develop hepatocellular adenoma and HCC owing to expression of the hepatitis B virus surface antigen. This whole-genome mutagenesis technique was used to generate an unbiased catalogue of candidate cancer genes (CCGs). Pooled shRNA libraries targeting 250 selected CCGs then were introduced into immortalized mouse liver cells and the cells were monitored for their tumor-forming ability after injection into nude mice.
Transposon-mediated mutagenesis identified 1917 high-confident CCGs and highlighted the importance of Ras signaling in the development of HCC. Subsequent pooled shRNA library screening of 250 selected CCGs validated 27 HCC tumor-suppressor genes. Individual shRNA knockdown of 4 of these genes (Acaa2, Hbs1l, Ralgapa2, and Ubr2) increased the proliferation of multiple human HCC cell lines in culture and accelerated the formation of xenograft tumors in nude mice. The ability of Ralgapa2 to promote HCC cell proliferation and tumor formation required its inhibition 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.
In a 2-step forward genetic screen in mice, we identified members of the Ral guanosine triphosphatase-activating protein pathway and other proteins as suppressors of HCC cell proliferation and tumor growth. These proteins might serve as therapeutic targets for liver cancer.
高通量测序技术已经在肝细胞癌（HCC）中鉴定出数千个罕见突变的基因。然而，由于肿瘤内和肿瘤间的高度异质性，加上大量的乘客突变，使得难以确定导致 HCC 发生的驱动突变。我们结合转座子诱变和小发夹 RNA（shRNA）文库的高通量筛选，以鉴定促进 HCC 发生的基因和通路。
转座子在表达乙型肝炎病毒表面抗原的转基因小鼠的肝脏中发生转座，这种全基因组诱变技术用于生成候选癌症基因（CCG）的无偏目录。然后将靶向 250 个选定 CCG 的 shRNA 文库池导入永生化的小鼠肝细胞，并在裸鼠中注射后监测其成瘤能力。
转座子介导的诱变鉴定出 1917 个高置信 CCG，并强调了 Ras 信号在 HCC 发展中的重要性。随后对 250 个选定 CCG 的 pooled shRNA 文库筛选验证了 27 个 HCC 肿瘤抑制基因。对这 4 个基因（Acaa2、Hbs1l、Ralgapa2 和 Ubr2）的单个 shRNA 敲低增加了多个人类 HCC 细胞系在培养中的增殖，并加速了裸鼠异种移植瘤的形成。Ralgapa2 促进 HCC 细胞增殖和肿瘤形成的能力需要其抑制 Rala 和 Ralb。使用小分子抑制剂 RBC8 和索拉非尼的组合对 Ras 信号进行双重抑制（通过 Ral 和 Raf），可降低 HCC 细胞在培养中的增殖，并完全抑制其在裸鼠中的异种移植瘤生长。
在小鼠的 2 步正向遗传筛选中，我们鉴定了 Ral 鸟嘌呤核苷酸三磷酸酶激活蛋白通路的成员和其他蛋白作为 HCC 细胞增殖和肿瘤生长的抑制因子。这些蛋白可能作为肝癌的治疗靶点。
