1] Cell Signaling and Developmental Biology Laboratory, Department of Biological Sciences, National University of Singapore, Singapore, Singapore [2] Mechanobiology Institute, National University of Singapore, Singapore, Singapore.
Molecular Biology Laboratory, Department of Biological Sciences, National University of Singapore, Singapore, Singapore.
Oncogene. 2014 May 22;33(21):2717-27. doi: 10.1038/onc.2013.240. Epub 2013 Jul 1.
RAS and Rho small GTPases are key molecular switches that control cell dynamics, cell growth and tissue development through their distinct signaling pathways. Although much has been learnt about their individual functions in both cell and animal models, the physiological and pathophysiological consequences of their signaling crosstalk in multi-cellular context in vivo remain largely unknown, especially in liver development and liver tumorigenesis. Furthermore, the roles of RhoA in RAS-mediated transformation and their crosstalk in vitro remain highly controversial. When challenged with carcinogens, zebrafish developed liver cancer that resembles the human liver cancer both molecularly and histopathologically. Capitalizing on the growing importance and relevance of zebrafish (Danio rerio) as an alternate cancer model, we have generated liver-specific, Tet-on-inducible transgenic lines expressing oncogenic Kras(G12V), RhoA, constitutively active RhoA(G14V) or dominant-negative RhoA(T19N). Double-transgenic lines expressing Kras(G12V) with one of the three RhoA genes were also generated. Based on quantitative bioimaging and molecular markers for genetic and signaling aberrations, we showed that the induced expression of oncogenic Kras during early development led to liver enlargement and hepatocyte proliferation, associated with elevated Erk phosphorylation, activation of Akt2 and modulation of its two downstream targets, p21Cip and S6 kinase. Such an increase in liver size and Akt2 expression was augmented by dominant-negative RhoA(T19N), but was abrogated by the constitutive-active RhoA(G14V). Consequently, induced expression of the oncogenic Kras in adult transgenic fish led to the development of hepatocellular carcinomas. Survival studies further revealed that the co-expression of dominant-negative RhoA(T19N) with oncogenic Kras increased the mortality rate compared with the other single or double-transgenic lines. This study provides evidence of the previously unappreciated signaling crosstalk between Kras and RhoA in regulating liver overgrowth and liver tumorigenesis. Our results also implicate that activating Rho could be beneficial to suppress the Kras-induced liver malignancies.
RAS 和 Rho 小 GTPases 是关键的分子开关，通过其独特的信号通路控制细胞动力学、细胞生长和组织发育。尽管在细胞和动物模型中已经了解了它们各自的功能，但它们在体内多细胞环境中的信号串扰的生理和病理生理后果在很大程度上仍然未知，特别是在肝脏发育和肝肿瘤发生中。此外，RhoA 在 RAS 介导的转化中的作用及其在体外的串扰仍然存在很大争议。当受到致癌物质的挑战时，斑马鱼会发展出类似于人类肝癌的肝癌，无论是在分子上还是组织病理学上。利用斑马鱼（Danio rerio）作为替代癌症模型的重要性和相关性不断增加，我们已经生成了肝脏特异性、Tet-on 诱导的转基因系，表达致癌性 Kras（G12V）、RhoA、组成型活性 RhoA（G14V）或显性负性 RhoA（T19N）。还生成了表达 Kras（G12V）和三种 RhoA 基因之一的双转基因系。基于定量生物成像和遗传和信号异常的分子标记，我们表明，早期发育过程中致癌性 Kras 的诱导表达导致肝脏增大和肝细胞增殖，伴随着 Erk 磷酸化的升高、Akt2 的激活及其两个下游靶标 p21Cip 和 S6 激酶的调节。这种肝脏大小的增加和 Akt2 表达的增加被显性负性 RhoA（T19N）增强，但被组成性活性 RhoA（G14V）阻断。因此，在成年转基因鱼中诱导表达致癌性 Kras 导致肝细胞癌的发展。生存研究进一步表明，与其他单或双转基因系相比，致癌性 Kras 与显性负性 RhoA（T19N）的共表达增加了死亡率。这项研究提供了以前未被认识到的 Kras 和 RhoA 之间在调节肝脏过度生长和肝肿瘤发生中的信号串扰的证据。我们的结果还表明，激活 Rho 可能有益于抑制 Kras 诱导的肝恶性肿瘤。
