The Age-Dependence and Cell-Specificity of Breast Cancer Driven by Mutant p53

项目摘要 乳腺癌是一种与年龄相关的疾病，老年女性发病率较高，年轻女性预后较差。 妇女大多数乳腺肿瘤是上皮起源的。乳腺上皮由外基底层组成， 细胞的肌上皮层和内腔细胞层。研究表明，细胞角蛋白 蛋白5（K5）标记产生基底细胞和腔细胞的双能祖细胞，和细胞角蛋白8 (K8)只产生管腔细胞的有标记的单能祖细胞。令人惊讶的是，尽管历史悠久， 乳腺癌研究、年龄如何影响乳腺癌以及乳腺肿瘤是否以及如何起源于K5 或K8阳性（K5+或K8+）祖细胞表现出的内在差异尚不清楚。回答这些问题 可能为改善诊断和个性化治疗提供新的途径， 病人。乳腺癌中最常见的遗传改变发生在肿瘤抑制基因p53中， 精氨酸248是顶部突变热点。新发表的条件性p53 wm-R245 W小鼠等位基因， 允许野生型p53转化为p53 R245 W（对应于人p53精氨酸至色氨酸突变 在密码子248处）响应Cre重组酶，是研究年龄依赖性的潜在有价值的工具 和突变型p53驱动的乳腺癌细胞特异性。作为未来机械和治疗的前奏 研究中，该试点项目将利用p53 wm-R245 W等位基因来建立乳腺癌模型并比较小鼠 乳腺肿瘤发生于不同年龄或不同细胞来源，这被假设表现为 在组织学、分子或基因组水平上的差异。这一假设将在三个具体目标中得到检验。 第一个目的是比较p53 R245 W突变体驱动的乳腺肿瘤在年轻和老年小鼠中开始。 将表达Cre的腺病毒（Ad-Cre）注射到2月龄或10月龄的乳腺导管中 雌性p53 wm-R245 W/+小鼠。肿瘤发生率、潜伏期和生长速率、动物存活率、转移和肿瘤的生长。 将在注射Ad-Cre的小鼠之间比较肿瘤的组织学和分子亚型的光谱 在各自的年龄。第二个目的是比较p53 R245 W驱动的乳腺肿瘤， K5+或K8+祖细胞。p53 wm-R245 W/+雌性小鼠将接受乳腺导管内注射 腺病毒AdK 5-Cre或AdK 8-Cre，其表达分别由K5或K8启动子引导的Cre。相同的 将在接受AdK 5-Cre或AdK 8-Cre的小鼠之间比较目标1中所述的参数。的 最后一个目的是在基因组水平上研究乳腺肿瘤的年龄依赖性和细胞特异性。 将对目的1和2中产生的肿瘤进行全外显子组测序（WES）。复发基因 将比较从2月龄和10月龄小鼠开始的乳腺肿瘤之间的变化， 以及在由K5+或K8+祖细胞起始的肿瘤之间。WES数据也将进行比较 人类乳腺癌测序数据。

PROJECT SUMMARY Breast cancer is an age-related disease, with higher incidence in elder women and worse prognosis in younger women. Most breast tumors are of epithelial origin. The mammary epithelium consists of an outer basal myoepithelial layer of cells and an inner luminal cell layer. Studies have described the existence of cytokeratin protein 5 (K5) marked bipotent progenitors giving rise to both basal and luminal cells, and cytokeratin protein 8 (K8) marked unipotent progenitors that produce luminal cells only. Surprisingly, despite the long history of breast cancer research, how age impacts breast cancer, and whether and how breast tumors initiated from K5 or K8 positive (K5+ or K8+) progenitors exhibit intrinsic differences remain unclear. Answering these questions may provide new avenues for improved diagnostics and personalized therapeutics that could prolong the lives of patients. The most frequent genetic alterations in breast cancer occur in the tumor suppressor p53, with arginine 248 being the top mutational hotspot. The newly published conditional p53wm-R245W mouse allele, which allows conversion of wild type p53 to p53R245W (corresponding to human p53 arginine to tryptophan mutation at codon 248) in response to Cre recombinase, is a potentially valuable tool for studying the age-dependence and cell-specificity of breast cancer driven by mutant p53. As a prelude for future mechanistic and therapeutic studies, this pilot project will utilize the p53wm-R245W allele to model breast cancer and compare mouse mammary tumors initiated at different ages or from different cellular origins, which are hypothesized to exhibit differences at the histological, molecular or genomic levels. This hypothesis will be tested in three specific aims. The first aim is to compare p53R245W mutant driven mammary tumors initiated in young and old mice. Adenoviruses expressing Cre (Ad-Cre), will be injected into the mammary ducts of 2 months- or 10 months old female p53wm-R245W/+ mice. Tumor incidence, latency and growth rate, animal survival, metastasis, and the spectra of histological and molecular subtypes of tumors will be compared between mice injected with Ad-Cre at the respective ages. The second aim is to compare p53R245W driven mammary tumors initiated from either K5+ or K8+ progenitor cells. p53wm-R245W/+ female mice will receive mammary intraductal injection of adenoviruses AdK5-Cre or AdK8-Cre that express Cre led by the K5 or K8 promoter, respectively. The same parameters as described in Aim 1 will be compared between the mice receiving AdK5-Cre or AdK8-Cre. The last aim is to investigate the age-dependence and cell-specificity of mammary tumors at the genomic level. Whole-exome sequencing (WES) will be performed on tumors produced in Aims 1 and 2. Genes with recurrent alterations will be compared between mammary tumors initiated from the 2 months- and 10 months old mice, as well as between tumors initiated from the K5+ or K8+ progenitor cells. The WES data will also be compared to the human breast cancer sequencing data.