Abstract 1579: Establishment of transgenic medaka as a stable tumor model for in vivo screening of anticancer drugs.

Abstract

Abstract The development of anticancer drugs would be facilitated by the availability of an in vivo screening system based on a multicellular organism. We intended to construct an animal model which can be stable and easily detected tumors without autopsy for chemical screening and assessment. In this study, the medaka which is a small egg-laying freshwater fish native to East Asia was applied. The medaka has proved suitable for toxicology and carcinogenesis in the past studies. Furthermore most of molecular technology is available to the medaka and the size of the medaka genome (∼800 Mb) is about half that of the zebrafish genome. To construct a tumor model using transgenic medaka fish, a promoter sequence suitable for the tissue specific tumorigenesis was selected. There are some genes having specific expression in melanocyte. Melanocyte specific expression of oncogenes may induce progression to melanoma. Therefore we selected a promoter sequence encoding Tyrosinase, one of the melanocyte specific genes. The recombinant plasmids for microinjection are consisting of the medaka tyrosinase promoter and human oncogenes (cMyc, BRAF mutant V600E and HRAS mutant G12V). One cell stage embryos were applied for microinjection. We obtained three stable transgenic individuals derived from one cMyc and two BRAF mutant (BRAF600E) injected F0, but none from HRAS mutant (HRAS12V). The transgenic (Tg)-cMyc and the Tg-BRAF600E produced tumor in internal organs with more than 50 % of all fish, but in eyes or skins (outer detectable organs) with less than 10 %. The Tg-HRAS12V animals died before achieving adulthood. We therefore developed a conditional gene expression system in which HRAS12V is expressed in response to the induction of Cre recombinase by heat treatment, given that the Cre gene was placed under the control of a medaka heat shock promoter. One of the stable transgenic lines developed abnormal pigment cell proliferation in the eyes and epidermis with 100% penetrance by 6 months postfertilization. Sorafenib, an inhibitor of RAS signaling, was administered to the transgenic fish and was found both to reduce the extent of melanophore proliferation and to improve survival. This Tg-HRAS12V medaka established here thus represents a promising in vivo system with which to screen potential anticancer drugs that target RAS signaling, and this system can readily be adapted for the screening of agents that target other oncogenes. Citation Format: Yuriko Matsuzaki, Haru Hosokai, Yukiyo Mizuguchi, Atsushi Shimizu, Hideyuki Saya. Establishment of transgenic medaka as a stable tumor model for in vivo screening of anticancer drugs. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1579. doi:10.1158/1538-7445.AM2013-1579