Minimal Myc functional threshold for tumorigenesis

我们设计了一种基因测试的假设，即需要最低水平的Myc功能才能“放大”促进和维持肿瘤形成所需的转录程序。我们使用小鼠p53缺失突变体(P53 KO)作为一个强大的肿瘤模型，通过引入内源性c-Myc水平(c-Myc+/-)的适度变化来测试Myc依赖性。比较c-Myc+/+(Myc WT；P53 KO)或c-Myc+/-(Myc-Het；P53 KO)基因相似的P53 KO同胞小鼠的无瘤存活时间。与Myc-WT相比，Myc-Het；P53 KO组的中位无瘤生存时间增加了一倍(P<0.0001)。这种差异与肿瘤类型无关，血管肉瘤和胸腺淋巴瘤是两组中最常见的肿瘤类型(83%)。用光谱核型分析、FISH、定量RNA原位杂交和免疫组织化学等方法分析了p53 KO肿瘤中c-Myc基因组的改变和表达水平，结果表明，在Myc-Het；P53 KO小鼠中，肿瘤发生了对最初减少的内源性c-Myc剂量的补偿。值得注意的是，在血管肉瘤中，通过几轮基因组四倍体获得的基因组扩增在Myc-Het；P53 KO肿瘤中始终高于Myc-WT；P53 KO肿瘤。虽然两种类型的胸腺淋巴瘤均未显示基因组扩增，但Myc-WT和Myc-Het肿瘤中Myc RNA和Myc蛋白的表达均较高，提示Myc基因剂量减少的代偿作用发生在转录水平。这些结果表明，减少内源性c-Myc剂量大大延缓了遗传上易发生肿瘤的小鼠的肿瘤发展，为了使Myc-Het；P53 KO小鼠发展成肿瘤，Myc表达的补偿性增加是必要的，这可以通过多种机制发生。我们还使用了携带条件c-Myc-Floated和他莫昔芬依赖的Cre等位基因的P53 KO小鼠胸腺淋巴瘤的同种异体移植，并使用双荧光报告程序来监测重组效果，以测试减少c-Myc是否会对已建立的肿瘤(即生长、进展)产生不利影响，并发现在从重组诱导的凋亡中恢复后，当内源性Myc剂量急剧减少时，肿瘤生长速度显著放缓。我们的结果强烈表明，Myc的适度减少可以抑制癌症的生长，并具有重要的意义，特别是对于延长Li-Fraumeni综合征患者(P53胚系突变)以及散发性癌症患者的无瘤生存期。这项研究为未来分析c-Myc在肿瘤发生和发展中的作用提供了一个框架和模型，并可以很容易地扩展到其他类型的肿瘤，使用类似的策略，以及其他肿瘤促进剂，如致癌RAS。我们还在探索是否在c-Myc表达水平严重降低或缺失的情况下，通过有选择地从出生后年轻小鼠的血管内皮组织(CDH5CreER)中同时去除p53和c-Myc来建立血管肉瘤模型。

We have designed a genetic test of the hypothesis that a minimum level of Myc function is required to "amplify" the transcriptional programs necessary for promoting and sustaining tumor formation. We employed the mouse p53 null mutant (p53 KO) as a robust tumor model to test for Myc-dependency by introducing modest changes in the endogenous c-Myc level (c-Myc+/-). Tumor-free survival times were compared in genetically similar p53 KO sibling mice that were either c-Myc+/+ (Myc WT; p53 KO), or c-Myc+/- (Myc-Het; p53 KO). Median tumor-free survival times doubled in the Myc-Het; p53 KO relative to Myc-WT (significant at P 0.0001). This difference was independent of the tumor type, with hemangiosarcoma and thymic lymphoma being the most common (83%) tumor types in both groups. Analyses of c-Myc genomic alterations and expression levels in p53 KO tumors, using Spectral Karyotyping (SKY), FISH, and quantitative RNA ISH and immunohistochemistry, revealed that compensation for the initially reduced endogenous c-Myc dosage had occurred in tumors arising in Myc-Het;p53 KO mice. Notably, in hemangiosarcomas, genome amplification achieved by several rounds of genome tetraploidization was consistently higher in the Myc-Het;p53 KO than in the Myc-WT;p53 KO tumors. Although thymic lymphomas of either genotype showed no genomic amplification, expression of Myc RNA and Myc protein were nevertheless comparably elevated in both the Myc-WT and Myc-Het tumors, suggesting that compensation for reduced Myc gene dosage had occurred at the transcriptional level. These results indicate that reduced endogenous c-Myc dosage substantially delays tumor development in mice that are genetically predisposed to neoplasia and that, in order for Myc-Het;p53 KO mice to develop tumors, a compensatory increase in expression of Myc, which can occur by multiple mechanisms, is required. We have also used allografts using of thymic lymphomas from p53 KO mice carrying conditional c-Myc-floxed and tamoxifen-dependent Cre alleles and a dual-fluorescent reporter to monitor recombination efficacy, in order to test whether reducing c-Myc will adversely impact established tumors (i.e. growth, progression) and found that after recovery from recombination induced apoptosis, tumor growth rate is substantially slowed when endogenous Myc dosage is acutely reduced. Our results strongly suggest that a modest reduction in Myc can curtail cancer growth and has important implications, particularly for extending tumor-free survival in patients with Li-Fraumeni syndrome (germline mutations in p53), as well as sporadic cancers. This study provides a framework and model for future analyses of c-Myc role in tumorigenesis and tumor progression and can be readily extended to other tumor types using analogous strategies, and other tumor promoters, such as oncogenic Ras. We are also exploring whether p53 tumors can arise at all in the context of severely reduced or absent c-Myc expression levels using a conditional knock-out approach to model hemangiosarcomas by removing both p53 and c-Myc selectively from endothelial tissues (Cdh5CreER) in post-natal young mice.