Modeling The Etiology of P53 Mutated Cancer Cells

描述（由申请人提供）：p53肿瘤抑制蛋白的失活被认为是大多数或所有人类癌症发展所必需的。超过50%的癌症具有破坏p53功能的TP 53突变。在大多数情况下，一个TP 53等位基因中的错义突变之后是杂合性缺失（洛），因此肿瘤仅表达突变的无活性形式的p53。在许多情况下，TP 53突变和洛缺失与治疗反应差、肿瘤侵袭性增加和长期生存率降低有关。尽管如此，对于TP 53点突变是如何获得的，洛缺失是如何发生的，或者涉及的细胞，我们知之甚少。大约15%的人类结直肠癌（CRC）是由于DNA错配修复（MMR）中的体细胞或遗传缺陷而导致的微卫星不稳定（MSI）。有趣的是，TP 53突变在MSI（MMR缺陷）CRC中的频率低于MMR完整的癌症。这表明MMR缺陷在MSI肿瘤发生中不会引起TP 53突变，尽管缺乏直接解决这个问题的实验。Wt p53由于MDM 2而以低水平表达，MDM 2是一种结合p53并促进其降解的E3连接酶。小分子MDM 2拮抗剂正被开发为治疗剂。Nutlin-3a（Nutlin）是一种MDM 2拮抗剂，通过阻断MDM 2-p53结合来稳定/激活p53。Nutlin在p53 wt细胞和人肿瘤异种移植物中引起稳健的生长停滞/凋亡。重要的是，只有p53-null或p53-突变体细胞可以在Nutlin中生长并形成集落。在这项资助计划中，我们利用Nutlin强大的生长抑制活性来选择p53突变的细胞，并研究TP 53突变是如何产生的以及洛缺失是如何发生的。初步数据表明，MMR缺陷型CRC细胞获得TP 53突变的速率高于修复熟练的对应物。此外，这些起始TP 53突变似乎在表达某些癌症干细胞（CSC）标志物（CD 44）的细胞中更普遍，而不是CSC标志物阴性细胞。最后，MSI细胞中的TP 53洛缺失通过拷贝中性机制发生。基于这些发现，我们假设1）MMR缺陷引起p53中的起始点突变，2）MSI细胞中的TP 53洛通过拷贝中性机制发生，以及3）CD 44（+）更倾向于获得TP 53突变，或者p53突变将CD 44低细胞转化为CD 44高细胞，从而可能增加癌症“干性”。

DESCRIPTION (provided by applicant): Inactivation of the p53 tumor suppressor protein is considered essential for the development of most or all human cancers. Over 50% of cancers harbor TP53 mutations that destroy p53 function. In most cases, missense mutations in one TP53 allele are followed by loss-of-heterozygosity (LOH), so tumors express only a mutant, inactive form of p53. TP53 mutations and LOH have been linked, in many cases, with poor therapy response, increased tumor aggressiveness, and decreased long-term survival. Despite this, remarkably little is known about how TP53 point mutations are acquired, how LOH occurs, or the cells involved. Approximately 15% of human colorectal cancers (CRCs) are microsatellite unstable (MSI) due to somatic or inherited defects in DNA mismatch repair (MMR). Interestingly, TP53 mutations are less frequent in MSI (MMR deficient) CRCs than cancers where MMR is intact. This has suggested MMR defects do not give rise to TP53 mutations in MSI tumorigenesis, though experiments to directly address this question are lacking. Wt p53 is expressed at low levels due to MDM2, an E3-ligase that binds p53 and promotes its degradation. Small molecule MDM2 antagonists are being developed as therapeutic agents. Nutlin-3a (Nutlin) is an MDM2 antagonist that stabilizes/activates p53 by blocking MDM2-p53 binding. Nutlin causes robust growth arrest/apoptosis in p53 wt cells and human tumor xenografts. Importantly, only p53-null or p53-mutant cells can grow and form colonies in Nutlin. In this grant proposal, we have leveraged the powerful growth inhibitory activity of Nutlin to select p53-mutated cells and examine how TP53 mutations arise and how LOH occurs. Preliminary data suggest MMR-deficient CRC cells acquire TP53 mutations at greater rate than repair-proficient counterparts. Further, these initiating TP53 mutations appear more prevalent in cells that express certain cancer stem cell (CSC) markers (CD44), but not CSC marker-negative cells. Finally, TP53 LOH in MSI cells occurs through a copy-neutral mechanism. Based on these findings we hypothesize 1) MMR deficiency gives rise to initiating point mutations in p53, 2) TP53 LOH in MSI cells occurs through a copy-neutral mechanism, and 3) CD44(+) are either more prone to acquire TP53 mutations, or p53 mutations convert CD44low cells into CD44hi and thus may increase cancer "stemness".