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的关键。超过50%的癌症含有破坏P53功能的TP53突变。在大多数情况下，一个TP53等位基因的错义突变会导致杂合性缺失(LOH)，因此肿瘤只表达一种突变的、不活跃的p53。在许多情况下，TP53突变和杂合性丢失有关，治疗反应差，肿瘤侵袭性增加，长期存活率降低。尽管如此，人们对TP53点突变是如何获得的，LOH是如何发生的，或者涉及的细胞知之甚少。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，在MSI(MMR缺陷)癌中，TP53突变的频率低于MMR完整的癌症。这表明MMR缺陷不会导致MSI肿瘤发生中的TP53突变，尽管缺乏直接解决这一问题的实验。WT P53的低水平表达是由于MDM2，一种结合P53并促进其降解的E3连接酶。小分子MDM2拮抗剂正被开发为治疗药物。Nutlin-3a(Nutlin)是一种MDM2拮抗剂，通过阻断MDM2-P53结合来稳定/激活P53。Nutlin可导致p53 wt细胞和人肿瘤异种移植瘤的生长停滞/凋亡。重要的是，只有p53缺失或p53突变的细胞才能在Nutlin中生长并形成克隆。在这项拨款提案中，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TP53突变是如何发生的，以及LOH是如何发生的。初步数据表明，缺乏MMR的CRC细胞获得TP53突变的速度比修复熟练的同类细胞更快。此外，这些启动的TP53突变似乎在表达某些癌症干细胞(CD44)标记的细胞中更为普遍，但不是CSC标记阴性的细胞。最后，MSI细胞中的TP53 LOH是通过复制中性机制发生的。基于这些发现，我们假设1)MMR缺陷引起P53的起始点突变，2)MSI细胞中的TP53 LOH通过拷贝中性机制发生，3)CD44(+)更容易获得TP53突变，或者P53突变将CD44low细胞转化为CD44hi，从而可能增加癌症的“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".