Minimal Myc functional threshold for tumorigenesis

我们设计了一种基因测试，假设最低水平的Myc功能需要“放大”促进和维持肿瘤形成所必需的转录程序。我们采用小鼠p53零突变体（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+/-）的适度变化来检测myc依赖性。在基因相似的p53 KO同胞小鼠中，比较c-Myc+/+ (Myc WT; p53 KO)或c-Myc+/- (Myc- het; p53 KO)的无肿瘤生存时间。Myc-Het的中位无瘤生存时间翻了一番；p53 KO相对于Myc-WT （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%）。利用光谱核型分析（SKY）、FISH、定量RNA ISH和免疫组织化学分析p53 KO肿瘤中c-Myc的基因组改变和表达水平，发现最初内源性c-Myc剂量减少的补偿发生在Myc-Het引起的肿瘤中；p53 KO小鼠。值得注意的是，在血管肉瘤中，通过几轮基因组四倍体化实现的基因组扩增在Myc-Het中始终较高；p53 KO在Myc-WT中较低；p53 KO肿瘤。尽管两种基因型的胸腺淋巴瘤均未出现基因组扩增，但Myc RNA和Myc蛋白的表达在Myc- wt和Myc- het肿瘤中均显著升高，这表明Myc基因剂量减少的补偿发生在转录水平。这些结果表明，内源性c-Myc剂量的减少大大延缓了遗传上易患肿瘤的小鼠的肿瘤发展，并且为了Myc-Het；p53 KO小鼠发生肿瘤，Myc表达的代偿性增加是必需的，这可以通过多种机制发生。我们还使用携带条件c-Myc-floxed和他莫昔芬依赖性Cre等位基因的p53 KO小鼠胸腺淋巴瘤异体移植物和双荧光报告基因来监测重组效果，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，并发现在重组诱导的细胞凋亡恢复后，内源性Myc剂量急剧减少时，肿瘤生长速度明显减慢。我们的研究结果强烈表明，适度减少Myc可以抑制癌症生长，并具有重要意义，特别是对于延长Li-Fraumeni综合征（p53种系突变）患者的无瘤生存期，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一个框架和模型，并且可以很容易地使用类似的策略扩展到其他肿瘤类型，以及其他肿瘤启动子，如致癌Ras。我们也在探索在c-Myc表达水平严重降低或缺失的情况下，p53肿瘤是否会出现，使用条件敲除方法，通过在出生后的年轻小鼠中选择性地从内皮组织（Cdh5CreER）中去除p53和c-Myc来模拟血管肉瘤。

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 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.