Minimal Myc functional threshold for tumorigenesis

我们设计了一个遗传学测试的假设，最低水平的Myc功能是必需的“放大”的转录程序，促进和维持肿瘤形成。我们采用小鼠p53无效突变体（p53 KO）作为稳健的肿瘤模型，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。在基因相似的p53 KO同胞小鼠中比较无肿瘤存活时间，所述同胞小鼠为c-Myc+/+（Myc WT; p53 KO）或c-Myc+/-（Myc-Het; p53 KO）。与Myc-WT相比，Myc-Het; p53 KO的中位无肿瘤生存时间增加了一倍（P <0.0001时具有显著性）。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%）。使用光谱核型分析（SKY）、FISH和定量RNA ISH和免疫组织化学分析p53 KO肿瘤中的c-Myc基因组改变和表达水平，结果显示，在Myc-Het;p53 KO小鼠中产生的肿瘤中发生了对最初降低的内源性c-Myc剂量的补偿。值得注意的是，在血管瘤中，通过几轮基因组四倍化实现的基因组扩增在Myc-Het;p53 KO中始终高于Myc-WT;p53 KO肿瘤。尽管两种基因型的胸腺淋巴瘤均未显示基因组扩增，但Myc RNA和Myc蛋白的表达在Myc-WT和Myc-Het肿瘤中均显著升高，表明在转录水平上发生了对Myc基因剂量减少的补偿。这些结果表明，降低内源性c-Myc剂量实质上延迟了遗传上易患瘤形成的小鼠中的肿瘤发展，并且为了使Myc-Het;p53 KO小鼠发展肿瘤，需要Myc表达的补偿性增加，这可以通过多种机制发生。我们还使用了来自p53 KO小鼠的胸腺淋巴瘤的同种异体移植物，该小鼠携带条件性c-Myc-floxed和他莫昔芬依赖性Cre等位基因，并使用双荧光报告基因监测重组效率，以测试减少c-Myc是否会对已建立的肿瘤产生不利影响（即生长、进展），并发现在从重组诱导的乳腺增生中恢复后，当内源性Myc剂量急剧减少时，肿瘤生长速率显著减慢。我们的研究结果强烈表明，Myc的适度减少可以减少癌症的生长，并具有重要的意义，特别是对于延长Li-Fraumeni综合征（p53生殖系突变）患者的无肿瘤生存期以及散发性癌症。该研究为未来分析c-Myc在肿瘤发生和肿瘤进展中的作用提供了框架和模型，并且可以使用类似策略轻松扩展到其他肿瘤类型以及其他肿瘤促进剂，例如致癌Ras。我们还在探索在c-Myc表达水平严重降低的情况下是否会出现p53肿瘤。

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 allelesand 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 apopstosis, 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 c-Myc expression levels.