Identifying colon cancer modifier genes with zebrafish

描述（由申请人提供）：在这个奖项申请中，我们概述了一种利用斑马鱼来识别调节癌细胞入侵的基因的策略，这是癌症转移的早期步骤。我们已经证明，斑马鱼熔解（mlt）突变体由于后肠上皮细胞的基质侵袭而产生癌症样表型，mlt突变体异位表达与癌症进展密切相关的人类基因的斑马鱼同源物。重要的是，抑制这些基因可以挽救侵袭性mlt表型。定位克隆鉴定出斑马鱼平滑肌肌球蛋白重链（MYH11）为mlt相关基因。人类MYH11也被证明是预测肿瘤转移的基因表达标志的一部分。总之，这些发现支持mlt作为一种侵袭性癌症模型。在这里，我们提出的实验将允许鉴定mlt修饰基因。这些基因的人类同源物被预测在癌症侵袭和转移中发挥作用，并可能成为抗癌治疗的靶点。设计了三组相互关联的实验来确定mlt修饰符。首先，我们将分析mlt突变体中斑马鱼同源基因的表达和功能，这些基因在癌细胞侵袭中起着确定的作用。其次，我们将比较mlt和兄弟野生型幼虫的转录谱，并通过反义敲除和强制表达实验评估差异表达基因的作用。第三，我们将进行化学诱变遗传筛选，以确定隐性和显性修饰因子的mlt癌样表型。总之，这些目标提供了一种识别调节人类癌症进展的基因的新方法。对mlt突变体中这些基因的功能评估提供了一种快速的、体内的药物靶向性测定方法。

DESCRIPTION (provided by applicant): In this award application we outline a strategy to identify genes that regulate cancer cell invasion, an early step in cancer metastasis, using the zebrafish. We have shown that zebrafish meltdown (mlt) mutants develop a cancer-like phenotype as a result of stromal invasion of posterior intestinal epithelial cells, mlt mutants ectopically express zebrafish orthologs of human genes strongly implicated in cancer progression. Importantly, inhibition of these genes rescues the invasive mlt phenotype. Positional cloning identified zebrafish smooth muscle myosin heavy chain (MYH11) as the responsible mlt gene. Human MYH11 has also been shown to be part of a gene expression signature that is predictive of tumor metastasis. Together, these findings support mlt as an invasive cancer model. Here, we propose experiments that will allow the identification of mlt modifier genes. The human orthologs of such genes are predicted to play a role in cancer invasion and metastasis, and may be targets for anti-cancer therapy. Three interrelated sets of experiments have been designed to identify mlt modifiers. First, we will analyze in mlt mutants the expression arid function of zebrafish orthologs of human genes with an established role in cancer cell invasion. Second, we will compare transcriptional profiles of mlt and sibling wild type larvae and assess the role of differentially expressed genes using antisense knockdown and forced expression experiments. Third, we will conduct a chemical mutagenesis genetic screen to identify recessive and dominant modifiers of the mlt cancer-like phenotype. Together, these aims provide a novel approach to identify genes that regulate human cancer progression. Functional assessment of these genes in mlt mutants provides a rapid, in vivo assay of their suitability for pharmacological targeting.